Vetiveria zizanioides, ext., acetylated

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Although a guideline study to GLP, heterogeneity between replicate results and non-dose-response related biological results were observed which may reflect the challenges of testing this UVCB substance using the WAF approach.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Item: Vetiveryl Acetate 112 Extra
Givaudan Code: 0519103
Batch Number: VE00464206
CAS Number: 84082-84-8
Content: complex mixture (UVCB)
Appearance: Yellow to brownish yellow liquid
Expiry date: January 07, 2019
Recommended storage: Dry, well ventilated, preferably full, hermetically sealed, ambient temperature (10 - 30 °C), protected against light

Analytical monitoring:

yes

Details on sampling:

The concentrations of the test item were analytically verified via GC-FID in fresh media at the start of the exposure and at renewal (0 and 24 hours) to check for consistent preparation of the WAFs and in old media at renewal and at the end of the test (24 and 48 hours) in all loading levels and the control to assess the stability of the WAFs during the test. Quantification was based on sum of the peak areas.

Preparation of the samples: 40 mL of the test item concentrations and the control were filled into a vial. 2 mL cyclohexane and 10% (w/w) sodium chloride were added. Afterwards the samples were shaken properly for 2 min (enrichment factor 20). 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 the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.

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 test items and UVCB substances, a modification of the standard method for the preparation of aqueous media was 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 / UVCB substance and is poorly soluble in water and in the permitted solvents. Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the test item phase was separated by siphon and the test organisms were 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. This preparation was repeated for water renewal after 24 hours.

Preparation of the water accommodated fractions:
For each loading level, a glass bottle with an appropriate capacity was filled up to the top with an appropriate amount of the dilution water leaving only a small headspace of a few milliliter. An appropriate volume of the test item (relative density: 0.9937 (20/20°C) was taken into account) was placed by pipette onto the water surface and the bottle was closed with the screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out for 48 hours with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 1 hour for separation of undissolved test item. Thereafter, the WAF was removed by siphoning from the approximate center of the water body. The WAFs were checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). Presence of undissolved test item during the test was observed and documented for all loading levels. The WAFs were prepared two days before the start of the exposure (day -2) and two days before the renewal of the test solutions (day -1) as described above

Loading levels:
Six water accommodated fractions (WAF) of the test item in a geometric series with a separation factor of 3 were prepared individually with dilution water (see Table 2) and were tested as follows:
0.2 -0.6 - 1.8 - 5.4 - 16.2 - 48.6 mg/L
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.
The test loadings are based on the results of a non-GLP preliminary range finding test. For results, see section 16.

Control
Dilution water without test item incubated under the same conditions as the test groups.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system: Daphnia magna STRAUS (Clone 5)

Reason for the selection of the test system: Daphnia magna is the preferred species in accordance with the test guidelines and is bred at the test facility.

Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Breeder: Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 +/- 2 °C, in an incubator, 16 hours illumination, light intensity of max. 1500 lx

Culture medium: Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaCO3/L, is used.

Feeding of the culture stocks: The daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae are cultured at the test facility.

Origin of the food algae: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used per WAF and control.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatization
Acclimatization of the daphnids was not necessary, because the dilution water was equivalent to the culture medium.

Application
The test vessels were filled up to the top with the test solutions. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette. At water renewal, the daphnids were taken from the old test vessels and directly transferred to the new ones. Thereafter, the test vessels were closed immediately with screw caps. There was no headspace in the test vessels.

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

48 h

Test temperature:

18 - 22 °C, constant within ± 1 °C

Nominal and measured concentrations:

Definitive test:
Nominal loading of the test item in the WAF: 48.6, 16.2, 5.4, 1.8, 0.6, 0.2 mg/L

The measured concentrations of the test item in fresh media at the start of the exposure (0 hours) were 0.175 – 0.369 – 0.620 – 0.918 – 1.92 – 4.42 mg/L and at renewal (24 hours) were
Per definition of the WAF, all terms related to concentration levels have to be given as loading levels because partly dissolved compounds and mixtures cannot be related to concentrations.

Details on test conditions:

Test temperature (target): 18 - 22 °C, constant within ± 1 °C
Illumination (target): Diffuse light, light intensity of max. 1500 lx
Photoperiod (target): 16/8 hours light/dark cycle
Feeding: The daphnids were not fed during the study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate p.a. (SIGMA)

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 48.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

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

The measured concentrations of the test item in fresh media at the start of the exposure (0 hours) were 0.175 - 0.369 - 0.620 - 0.918 - 1.92 - 4.42 mg/L and at renewal (24 hours) were
The biological results observed were non-dose related. The mean % immobilization was 0% (control), 5% (0.2mg/L loading level), 30% (0.6 mg/L loading level), 30% (1.80 mg/L loading level), 10% (5.4 mg/L loading level), 15% (16.2 mg/L loading level) and 0 % (48.6 mg/L loading level). As a consequence no statistical analysis could be carried out. However, given that the mobility observed was <=30% at all loading rates, it was concluded that the EL50 was greater than the highest concentration tested which was a loading rate of 48.6 mg/L.

Differences between the four replicates were observed for most treatments:
Loading rate 48.6 mg/L: 0, 0, 0, 0%
Loading rate 16.2 mg/L: 0, 60, 0, 0 %
Loading rate 5.40 mg/L: 0, 0, 40, 0%
Loading rate 1.8 mg/L: 20, 100, 0, 0%
Loading rate 0.6 mg/L: 100, 20, 0, 0%
Loading rate 0.2 mg/L: 20, 0, 0, 0%
Control: 0, 0, 0, 0%

All WAFs were visually clear throughout the exposure period but did show a positive Tyndall effect at the start of the exposure and renewal. This could indicate the presence of undissolved test item in the WAFs, that perhaps played a part in the different effects seen between replicates. This was not observed in the algae study performed at the same test facility using WAFs and perhaps reflects a higher sensitivity of daphnia to artifacts such as undissolved test item. Another possible explanation provided in the report is that different compounds of the UVCB test item may have affected the daphnia in different ways.

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2017-11-02 to 2017-11-03. EC50 = 2.10 mg/L, 95% confidence limits 1.92 - 2.43 mg/L

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal loadings of the test item VETIVERYL ACETATE 112 EXTRA, the 48 hour-EL50 for Daphnia magna was > 48.6 mg/L.

Due to the non-dose-response related biological effects, all effect values were determined empirically. No statistical evaluation was carried out.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Although a guideline study to GLP, there was no analytical verification of test item concentrations at test end.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

The test item was only analytically verified at test begin.

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Item: Vetiveryl Acetate Extra
Batch Number: 20070028
Appearance: colourless to pale yellowish liquid
Expiry date: 2001-03-31
Recommended storage: Keep in a cool and dry place
Storage at test facility: Refrigerator 7+/- 2°C, protected from light and moisture

Analytical monitoring:

yes

Details on sampling:

DOC was measured in all concentration levels and control at test start (Oh, fresh media).

Vehicle:

no

Details on test solutions:

Dispersion treatment 24 h on a magnetic stirrer
Stock solution: Saturated solution (130 mg/L were weighed out) prepared with dilution water one day prior to application and filtered before use
(0.45 pm).
Test dilutions: 1:16, 1:8, 1:4, 1:2, 1:1
Test concentrations 1.5 — 3.0 — 6.8 — 14.8 — 39 mg/L (based on DOC-analysis)
CONTROL: Dilution water without test item tested under the same conditions as the test groups.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST SYSTEM Daphnia magna STRAUS (Clone 5)
Reason for the selection: Daphnia magna STRAUS belongs, in accordance with the study guideline, to the suitable Daphnia types and are bred in the test facility.
Origin: Institut für Wasser-, Boden- und Lufthygiene des Bundesgesundheitsamtes, Corrensplatz, D-14195 Berlin
Breeder: DR.U.NOACK-LABORATORIUM, D-31157 Sarstedt
Culture: In 2-3 L glass vessels with approximatively 1.8 L culture medium, by 21 °C (temperatures of 20-25 °C were tolerated), in an incubator, 16 h illumination, illumination strength 1.5-5.0 pmol•m-2-s-1.
Culture medium: Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.
Feeding: 5 x weekly ad libitum with a mix of Scenedesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 106 cells/mL.

Number of study organisms per concentration and control: 20 animals, divided into 4 parallel samples, each with 5 animals
Number of animals per test vessel: 5
Number of parallel samples per concentration: 4
Age of test organisms: Less than 24 h old
Illumination: Diffuse light, illumination range 1.5 - 5 umol•m-2 • s-1
Photoperiod: 16/8 h light/dark cycle

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

Test temperature 21 ± 1 °C constant

Nominal and measured concentrations:

Definitive Test
Measured concentrations at test start: 39, 14.8, 6.8, 3.0 and 1.5 mg/L (calculated based on results of DOC)

Details on test conditions:

TEST DESCRIPTION
The preliminary range finding test was conducted under static conditions with three dilution of a stock solution (1:1, 1:10, 1:100, NON-GLP-state). For the stock solution 100 mg/L were weighed out. The test solution was stirred 24 h and filtered before use.

Based an the resuits of preliminary tests a definitive test was performed with 5 dilutions of the saturated stock solution (130 mg/L) ranging from 1:1 to 1:16 with a dilution factor of 2 to enable the determination of 0 and 100 % immobilisation after 24 and 48 hours.

2 to 24 h old daphnids were used for the preliminary and definitive test. They had been obtained by removing the mother animals twice within 22 h. The daphnids had been acclimatized to the dilution water 2 h before test started under test conditions.

20 daphnids divided into 4 (2*) groups with 5 (10*) animals per test vessel were used. Control replicates without test item were tested under the same conditions as the test groups. All test beakers were placed in an incubator (light/dark cycle 16/8 for 48 h at 21 ± 1 °C).
In the preliminary and in the definitive test the immobilisation of the daphnia was observed after 24 and 48 hours of exposure.

The reference item was tested in five concentrations within the • concentration range 0.58 to 5.8 mg/L.

Observations, measurements and evaluation were carried out as described below.

TYPE AND FREQUENCY OF MEASUREMENTS AND OBSERVATIONS
Prior to test start pH-values, dissolved oxygen concentration, conductivity, total hardness and temperature of the dilution water were measured.
At the beginning of the test water parameters (pH value, oxygen concentration) were measured in one additional replicate per concentration and control groups. After 48 h the water parameters in the old media were measured in all replicates per concentration and control.
The temperature was recorded throughout the test with a thermohygrograph.
The percentage immobility was determined in all test and control groups after 24 h and 48 h.

CONCENTRATION CONTROL ANALYSIS
The test item was analytically verified via DOC analysis according to DIN 38409 part 3.
DOC was measured in all concentration levels and control at test start (0h, fresh media).

EQUIPMENT
Oxygen : Oximeter, Oxi 191 (WTW)
pH value : pH-Meter 537 (WTW)
Temperature : Hygro-thermograph (KLIMATHERM)
Water hardness : Spectrophotometer, Cadas 100 LPG 158 (DR. LANGE)
Fluorescence of algae : Chlorophyll-Fluorometer (KLEINFELD)
DOC-analysator : MUlti N/C, ANALYTIK JENA

Reference substance (positive control):

yes

Remarks:

potassium dichromate p.a. (MERCK) (CAS No. 7778-50-9)

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

5.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

7.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL 6.6-7.8

Details on results:

The water parameters (dissolved oxygen concentration, pH-value) of test and control groups at the beginning of the test were measured in one replicate per concentration and control (Table 4). After 48 h water parameters of all replicates of the old media were measured.
pH-value, oxygen concentration, temperature, conductivity and total hardness of the dilution water were measured prior to test start.
DOC was measured in the new media (0 h) of all test concentrations and control.
Real concentrations were calculated based an determined DOC analysis.
During the test the temperature was in the range 21 ± 1 °C.

Validity Criteria
• In the control group no Daphnia were immobilised or trapped an the surface of the water.
• The EC50 - value after 24 h of the reference item was in the range of 1.0 to 2.5 mg/L.
• The dissolved O2 concentration was > 5.21 mg/L (60% of the air saturation value at the temperature used).
• The pH-value did not vary by more than 1 unit.

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 h. The EC100 value was determined directly from the test results. The EC10 and EC50 with 95 % confidence interval (CI) was determined in a probability network by interpolation according to standard procedures. EC- values after 24 h of the reference item in [mg/L]:
EC10 : 1.3
EC50 : 2.0 (CL 1.8 - 2.2)
EC100 : 5.8

The EC50 - value of reference item potassium dichromate after 24 h is within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS: DIN Guideline 38412 L 30.

Validity criteria fulfilled:

yes

Conclusions:

The acute immobilisation (EC50) of the test item VETIVERYLACETATE EXTRA batch number 20070028, to Daphnia magna (STRAUS) was determined according to OECD Guideline 202 I/ EEC Directive 92/69/EEC Method C.2.
The 48h-EC10 was calculated to be 5.6 mg/L and the 48-EC50 was calculated to be 7.2 mg/L (95% CL 6.6-7.8) based on initial measured concentrations.

Description of key information

The acute toxicity of Vetiveryl Acetate to daphnia magna has been investigated in two independent OECD 202 studies over a duration of 48 hours (Noack 2001, Klix 2018).

The first study was conducted under static conditions and the test item solutions prepared by dilution of a saturated solution prepared at a loading rate of 130mg/L (Noack 2001). The test dilutions were 1:16, 1:8, 1:4, 1:2 and 1:1. The actual item concentrations were measured at the start of test using DOC analysis and were respectively 1.5, 3.0, 6.8, 14.8 and 39 mg/L. A dose response was observed with the mean % immobilisation being respectively 0 (control), 0, 10, 40, 100 and 100%. The 48h EC50 was determined to be 7.2mg/L with 95% confidence levels of 6.6 to 7.8 mg/L. All validity criteria were met. The study is rated reliability 2 (reliable with restrictions) due to a number of deficiences, i.e. no analysis at test end despite the potential volatile properties of test item components and use of a static design.

A new test was conducted using a semi-static design with renewal of test solutions after 24 hours to maximise exposure over the duration of the test and using sealed glass flasks without headspace to miminise loss of test item by evaporation (Klix 2018). Given that Vetivery acetate is a UVCB substance comprising components that are not fully soluble in water, the test item was prepared as a water accommodated fraction (WAF). Six WAFs were prepared at loading rates of 0.200 - 0.600 - 1.80 - 5.40 - 16.2 - 48.6 mg/L. The concentrations of the test item were analytically verified via GC-FID in fresh media at the start of the exposure and at renewal (0 and 24 hours) to check for consistent preparation of the WAFs and in old media at renewal and at the end of the test (24 and 48 hours) in all loading levels and the control to assess the stability of the WAFs during the test. Quantification was based on sum of the peak areas.

The measured concentrations of the test item in fresh media at the start of the exposure (0 hours) were 0.175 - 0.369 - 0.620 - 0.918 - 1.92 - 4.42 mg/L and at renewal (24 hours) were <LOQ - 0.243 - <LOQ - 0.657 - 1.13 - 3.01 mg/L. The expected trend of increasing concentration with increasing loading rate was observed. The measured concentrations in the fresh media (0 and 24 hours) were slightly different for a given loading level, but in view of the low solubility and expected volatility of the test item this is still considered as consistent preparation of each WAF. The measured concentrations in old media were 54 to 79% (24h old) and 69 to 100% (48h old) of the initially measured concentrations indicating some instability of WAF components. The geometric mean measured concentrations were 0.115 - 0.251 - 0.235 - 0.665 - 1.26 - 3.24 mg/L which when compared to the initial concentration indicates that exposure over the 48 hour period was reasonable well maintained.

Test data obtained with WAFs apply to the multi-component substances as an entity because partly dissolved compounds and mixtures cannot be related to concentrations.

Therefore, all terms related to concentration levels are given as loading levels The biological results were non-dose related. The mean % immobilization was 0%, 5%, 30%, 30%, 10%, 15% and 0% for respectively the control and loading rates of 0.200 - 0.600 - 1.80 - 5.40 - 16.2 - 48.6 mg/L. As a consequence no statistical analysis could be carried out. However, given that the mobility observed was <=30% at all loading rates, it was concluded that the EL50 was greater than the highest concentration tested which was a loading rate of 48.6 mg/L.

Some differences were observed in the % immobility between replicates at most treatment levels. Thus the study has been assigned a reliability of 2 (reliable with restrictions). All WAFs were visually clear throughout the exposure period but did show a positive Tyndall effect at the start of the exposure and renewal. This could indicate the presence of undissolved test item in the WAFs, which perhaps played a part in the different effects seen between replicates. This was not observed in the algae study performed at the same test facility using WAFs and perhaps reflects a higher sensitivity of daphnia to artifacts such as undissolved test item.

Despite the heterogeneity between replicates, the results from the second study (Klix 2018) where exposure was reasonably well maintained over the 48 hours, show an EL50 of > 48.6 mg/L. For complex mixtures such as Vetivery Acetate the acute loading level from aquatic toxicity tests using WAFs may be used directly for classification. However, taking a worst-case approach the EC50 of 7.2 mg/L based on initial measured concentrations obtained from the first study (Noack 2001) where dilutions of a saturated solution were applied has been selected as the key study for the purpose of classification and labelling.