Vetiveria zizanioides, ext.

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 April 2010 - 13 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles. The validity criteria were fulfilled.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Vetiveria zizanioides, ext.

EC Number:

282-490-8

EC Name:

Vetiveria zizanioides, ext.

Cas Number:

84238-29-9

Molecular formula:

Not available due to the UVCB nature of the substance

IUPAC Name:

Essential oil of Vetiveria zizanioides (Poaceaes)

Constituent 2

Reference substance name:

Vetiveria zizanioides, ext.

IUPAC Name:

Vetiveria zizanioides, ext.

Details on test material:

- Name of test material (as cited in study report):Vetiveria zizanioides, ext.
- Substance type:Clear brown liquid (determined at NOTOX)
- Physical state: Liquid
- Analytical purity:Not indicated by the sponsor
- Lot/batch No.:ADL 53 901
- Stability under storage conditions: Stable
- Storage condition of test material:At room temperature in the dark
- Expiry date: 04 January 2011
- Stability in water: Unknown
- Solubility in water: 10-20 mg/mL (estimated)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not relevant

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

During the final test singular samples for possible TOC-analysis were taken from all WAFs and the control according to the schedule below.
Frequency: at t=0 h and t=48 h
Volume : 40 ml
Storage : Samples were stored in a refrigerator until analysis.
At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Test solutions

Vehicle:

no

Details on test solutions:

All test solutions were individually prepared by mixing exact amounts of test substance with test medium. These aqueous mixtures were then magnetically stirred for 48 hours in closed vessels in the dark. Thereafter, all solutions were left to stabilise overnight. This resulted in generally clear solutions (except the 100 mg/l that was slightly hazy) containing a floating layer. Subsequently, the Water Accommodated Fractions (WAFs) were collected by siphoning the water phase into the test vessels. The final test solutions were all clear and colourless, except for the WAF prepared at 100 mg/l that was slightly hazy.
The oily substance consists of a mixture of components with different solubility and behaviour in water. Some of these components might be volatile and/or light sensitive as indicated by the sponsor. As a consequence, all handling and weighing were performed under yellow light or dimmed light conditions and using closed systems with limited headspace.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: In-house laboratory culture
- Age at study initiation (mean and range, SD): < 24 hours
- Method of breeding: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
- Feeding during test: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

not included

Test conditions

Hardness:

180 mg/l expressed as CaCO3

Test temperature:

between 20.1 and 20.9°C

pH:

At t=0 h: 7.6
At t= 48 h: 7.5 - 7.6

Dissolved oxygen:

At t=0 h: 8.6 – 8.9
At t= 48 h: 8.8 - 9.0

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations:
WAFs prepared at loading rates of 0.46, 1.0, 2.2, 4.6 and 10 mg/l.

Measured concentrations:
TOC-analysis showed that recoveries for organic carbon remained fairly stable during the test period. The average measured exposure concentrations were calculated to correspond to 0.14, 0.64, 1.8 and 4.5 mg/l and thus represented 14, 29, 39 and 45% relative to the loading rates of 1.0, 2.2, 4.6 and 10 mg/l, respectively. It should be noted that Carbon measurements at concentrations close to or below 1 mg/l have to be considered estimates due to the limited sensitivity of the equipment.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 ml, all glass
- Type (delete if not applicable): closed with a screwcap - ~40 ml, normal headspace
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium
- Culture medium different from test medium: yes, for culturing M7 medium is used

OTHER TEST CONDITIONS
- Adjustment of pH: 7.7 ± 0.3
- Photoperiod: 16 hours photoperiod daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : EC50 daily

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Test concentrations: WAFs prepared at loading rates of 0.46, 1.0, 2.2, 4.6 and 10 mg/l.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

1.9

Nominal / measured:

meas. (initial)

Conc. based on:

dissolved

Remarks:

(Total Organic Carbon (TOC) converted to test material)

Basis for effect:

mobility

Remarks on result:

other: 95% CI 1.4-2.5 mg/l

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.95 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

dissolved

Remarks:

(Total Organic Carbon (TOC) converted to test material)

Basis for effect:

mobility

Remarks on result:

other: 95% CI 0.68 - 1.4 mg/l

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.14

Nominal / measured:

meas. (initial)

Conc. based on:

dissolved

Remarks:

(Total Organic Carbon (TOC) converted to test material)

Basis for effect:

mobility

Effect concentrations 4

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

4.9 other: mg/l (Loading rate)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI 4.0-6.1 mg/l

Effect concentrations 5

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.6 other: mg/l (Loading rate)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI 2.0 - 3.9 mg/l

Effect concentrations 6

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1 other: mg/l (Loading rate)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

behavioural abnormalities: no
- Mortality of control: 5% immobility
- Other adverse effects control: no

Results with reference substance (positive control):

Results with reference substance valid? yes
- EC50/LC50:
The 24h-EC50 was 0.75 mg/l with a 95% confidence interval between 0.56 and 1.0 mg/l.
The 48h-EC50 was 0.36 mg/l with a 95% confidence interval between 0.28 and 0.49 mg/l.

Reported statistics and error estimates:

The EC50-value was calculated at 24 and 48 hours of exposure from the probits of the percentages of affected daphnids and the logarithms of the corresponding test substance concentrations using the maximum likelihood estimation method (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition).

Any other information on results incl. tables

Acute immobilisation of daphnids after 24 and 48 hours in the final test

Test group Vetiveria zizanioides, ext.		Vessel number	Number Daphnia exposed	Response at 24 h		Response at 48 h
WAF prepared at x mg/l	Estimated average conc. (mg/l)			number	Total %	number	Total %
Control	-	A  B C D	5 5 5 5	0 0 0 0	0	0 1 0 0	5
0.46	n.m.	A  B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
1.0	0.14	A  B C D	5 5 5 5	0 0 0 1	5	0 0 0 1	5
2.2	0.64	A  B C D	5 5 5 5	0 1 0 0	5	1 2 0 0	15
4.6	1.8	A  B C D	5 5 5 5	1 2 2 3	40	4 5 5 5	95
10	4.5	A  B C D	5 5 5 5	5 (1)  4 5 (1) 5	95	5 5 5 5	100

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

n.m. Not measured

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study Vetiveria zizanioides, ext. did not induce significant acute immobilisation of Daphnia magna at a WAF prepared at a loading rate of 1.0 mg/l after 48 hours of exposure (NOEC). The average exposure concentration based on TOC-analyses was estimated at 0.14 mg/l.

The 48h-EC50 was 0.95 mg/l based on average exposure concentrations (95% confidence interval between 0.68 and 1.4 mg/l ). When based on loading rates, the 48h-EL50 corresponded to 2.6 mg/l with a 95% confidence interval between 2.0 and 3.9 mg/l.

Executive summary:

The acute toxicity of Vetiveria zizanioides, ext. was tested against Daphnia magna according to OECD guideline No. 202, 2004. In view of the oily character, the test solutions were prepared as Water Accomodated Fractions (WAF), by magnetic stirring, settlement and subsequent collection of the soluble fraction. WAFs were prepared at loading rates of 0.46, 1.0, 2.2, 4.6 and 10 mg/l. The total test period was 48 hours.

Samples were taken for analyses of Total Organic Carbon (TOC) concentrations at the start and the end of the 48-hour test period. No specific analysis was possible due to the composition of the test substance (oily mixture). The average measured exposure concentrations were calculated to correspond to 0.14, 0.64, 1.8 and 4.5 mg/l and thus represented 14, 29, 39 and 45% relative to the loading rates of 1.0, 2.2, 4.6 and 10 mg/l, respectively. The lowest WAF of 0.46 mg/l was not measured.

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

Vetiveria zizanioides, ext. did not induce significant acute immobilisation of Daphnia magna at a WAF prepared at a loading rate of 1.0 mg/l after 48 hours of exposure (NOEC). The average exposure concentration based on TOC-analyses was estimated at 0.14 mg/l. The 48h-EC₅₀was 0.95 mg/l based on average exposure concentrations (95% confidence interval between 0.68 and 1.4 mg/l[FBA1] ). When based on loading rates, the 48h-EL₅₀corresponded to 2.6 mg/l with a 95% confidence interval between 2.0 and 3.9 mg/l.