Registration Dossier

Ecotoxicological information

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:
06-04-2010 - 31-05-2010
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

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)
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
- Expiration date of the lot/batch:04 January 2011
- Stability under storage conditions:Stable
- Storage condition of test material:At room temperature in the dark
-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 (from vessels incubated without algae) 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.

Test solutions

Vehicle:
no
Details on test solutions:
The batch of Vetiveria zizanioides, ext. tested was a clear brown liquid that was not completely soluble in test medium at the concentrations tested. 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.

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 32 and 100 mg/l that were 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 WAFs prepared at 32 and 100 mg/l that were very slightly hazy.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 3 or 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10E4 cells/ml.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
0.24 mmol/l (24 mg CaCO3/l)
Test temperature:
between 21.4 and 22.1°C
pH:
At t=0 h: 7.3 - 7.4
At t=72 h: 7.5 - 9.4
Nominal and measured concentrations:
Nominal concentrations: WAF prepared at a loading rate of 0.32- 1.0 - 3.2 - 10 - 32 and 100 mg/l
Measured concentrations:
TOC-analysis showed that recoveries for organic carbon remained stable during the test period. The average measured exposure concentrations were calculated to correspond to 0.30, 0.63, 1.9, 4.8, 11.8 and 23.9 mg/l and thus represented 94, 63, 59, 48, 37 and 24% relative to the loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 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: 100 ml, all-glass, closed with a septum, containing 75 ml of test solution
- Type: airtight closed test vessels
- Material, size, headspace, fill volume: 100 ml, normal headspace, 75 ml
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: 298000 cells/ml
- No. of vessels per concentration (replicates): 3 replicates of each test concentration + 1 replicate of each test concentration without algae.
- No. of vessels per control (replicates):6 replicates



GROWTH MEDIUM
Adjusted M2, i.e. based on the OECD 201 Guideline but with a double amount of NaHCO3. In addition, pH is adjusted to 7. Adjusted M2 is formulated using Milli-Q water preventing precipitation

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: pH is adjusted at the start of the study (pH 7.0)
- Photoperiod: continuous
- Light intensity and quality: TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 87 to 93 E.m-2.s-1.



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with cuvettes (pathlength =10 mm). Algal medium was used as blank and the extra replicates without algae as background for the treated solutions.]
- Other: 48 h NOErC, 48 h NOEbC, 48 h ErC50, 48 h EbC50


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Test concentrations: WAF’s prepared at a loading rate of 0 - 0.32 -1.0 - 3.2 - 10 - 32 - 100 mg/l
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.3 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Remarks:
Total Organic Carbon (TOC) converted to test material
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.32 other: mg/l (Loading rate)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
1.8 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Remarks:
Total Organic Carbon (TOC) converted to test material
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 1.2-2.8 mg/l
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
3 other: mg/l (Loading rate)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 1.7-5.1 mg/l
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
9.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Remarks:
Total Organic Carbon (TOC) convereted to test material
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 5.9-14 mg/l
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
23 other: mg/l (Loading rate)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 14-38 mg/l
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.3 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Remarks:
Total Organic Carbon (TOC) converted to test material
Basis for effect:
other: Yield
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.32 other: mg/l (Loading rate)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0.4 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Remarks:
Total Organic Carbon (TOC) convereted to test material
Basis for effect:
other: Yield
Remarks on result:
other: 95% CI 0.21-0.74 mg/l
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0.49 other: mg/l (Loading rate)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95% CI 0.22-1.1 mg/l
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
2.4 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Remarks:
Total Organic Carbon (TOC) converted to test material
Basis for effect:
other: Yield
Remarks on result:
other: 95% CI 2.0- 9.4 mg/l
Details on results:
Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
Results with reference substance (positive control):
Potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/l and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) was 1.2 mg/l with a 95% confidence interval ranging from 0.77 to 2.0 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the present batch corresponds with this range.

The EC50 for yield inhibition (EYC50: 0-72h) was 0.44 mg/l with a 95% confidence interval ranging from 0.37 to 0.54 mg/l. The historical ranges of the 72h EC50 for yield inhibition lie between 0.43 and 1.1 mg/l. Hence, the EYC50: 0-72h for the present batch corresponds with this range.
Reported statistics and error estimates:
Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test substance.

Growth rates gradually decreased with increasing concentrations during the 48-hour test period. Statistically significant reduction of growth rate was found at average exposure concentrations of 0.63 mg/l and higher (Bonferroni t test, α = 0.05).

Inhibition of yield increased with increasing concentration of Vetiveria zizanioides, ext. resulting in more than 90% inhibition at the highest test concentration. Statistically significant inhibition of yield was found at average exposure concentrations of 0.63 mg/l and higher (Bonferroni t test,
α = 0.05).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, Vetiveria zizanioides, the 48h-EL50 corresponded to 23 mg/l with a 95% confidence interval ranging from 14 to 38 mg/l.
Executive summary:

Under the conditions of the present study with Pseudokirchneriella subcapitata, Vetiveria zizanioides, ext. reduced growth rate and inhibited the yield of this fresh water algae species significantly at a WAF prepared at a loading rate of 1.0 mg/l, corresponding to an average measured concentration of 0.63 mg/l.

The EC50 for growth rate reduction (ERC50: 0-48h) based on average measured concentrations was 9.1 mg/l with a 95% confidence interval ranging from 5.9 to 14 mg/l. When based on loading rates the EL50 corresponded to 23 mg/l with a 95% confidence interval ranging from 14 to 38 mg/l.

The EC50 for yield inhibition (EYC50: 0-48h) based on average measured concentrations was

2.4 mg/l with a 95% confidence interval ranging from 1.3 to 4.3 mg/l. When based on loading rates the EL50 corresponded to 4.4 mg/l with a 95% confidence interval ranging from 2.0 to 9.4 mg/l.

The NOEC for both growth rate reduction and yield inhibition was 0.30 mg/l based on measured concentrations. The NOEL, based on loading rates, corresponded to a WAF prepared at 0.32 mg/l.