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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Jan 2018 - 27 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Adopted March 23, 2006; Annex 5 corrected 28 July 2011.
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23
Version / remarks:
2000
Deviations:
no
Principles of method if other than guideline:
Samples for Total Organic Carbon (TOC) analysis were taken from all WAFs prepared at 0.80 mg/L and higher and from the control. Replicate vessels for TOC analysis were prepared only for the control and WAFs at 0.80 mg/L and higher
GLP compliance:
yes
Specific details on test material used for the study:
Identification: Ylang Ylang III
Appearance: Pale yellow clear liquid
Batch: 17106567
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 30 November 2019 (expiry date)

Additional information:
Test Facility test item number: 208293/C
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name (IUPAC, synonym or trade name: Ylang Ylang III oil
CAS number: 83863-30-3 (8006-81-3)
Irritant or corrosive: Yes
Specific gravity / density: 0.914
Analytical monitoring:
yes
Remarks:
TOC measurement
Details on sampling:
The extra replicates of the control and the WAFs at 0.80 mg/L and higher but without algae and without HEPES were used for possible analysis according to the schedule below.
Frequency: at t=0 h and t=72 h
Volume: 40 mL
Storage: Samples were stored in a refrigerator (2-8°C) until analysis

Additionally, reserve samples of 40 mL were taken from the control and the WAFs at 0.80 mg/L and higher but without algae and without HEPES for possible analysis. If not already used, these samples were stored in a refrigerator (2-8°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Remarks:
Water Accomodated Fractions (WAFs) were used
Details on test solutions:
The batch of Ylang Ylang III tested was a pale yellow clear liquid. The test item was a UVCB and not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item. During preparation of test solutions the test item was treated as being possibly volatile.
Preparation of test solutions started with loading rates individually prepared at concentrations ranging from 0.10 to 100 mg/L. A two-day period of magnetic stirring in closed vessels with minimal headspace in the dark was applied to ensure maximum dissolution of the test item in medium. The obtained aqueous mixtures were allowed to settle overnight. Thereafter, the Water Accommodated Fractions (WAFs) were collected by means of siphoning and microscopically inspected for the presence of undissolved test material. For the final test two additional test concentrations were prepared by subsequent dilution of the lowest loading rate in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

After preparation, volumes of 40 mL were added to each replicate vessel of the respective test concentration containing a 0.8 mL of an algal suspension providing a cell density of 10^4 cells/mL.Any residual volumes were discarded.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)
Strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Stock culture : 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.
Pre-culture: 3 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 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
During the exposure period the temperature measured in the incubator varied between 22 and 24°C.
pH:
t=0h : 7.3 - 7.6
t =72h: 7.4 - 7.9
The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 unit).
Nominal and measured concentrations:
Nominal: WAFs at 0.16, 0.80, 4.0, 20 and 100 mg/L. In addition, 4% and 20% of the WAF at 0.16 mg/L. These latter two concentrations are further indicated as WAFs at 0.0064 and 0.032 mg/L

TOC measurements corrected for the control treatment:
Loading rates: Control, 0.80, 4.0, 20 and 100 mg/L
TOC t=0h : n.a, n.q, n.q, n.q and 5.6 mg/L
TOC t=72h: n.a, n.q, n.q, n.q and 3.1 mg/L

n.a. – not applicable, n.q. – not quantifiable

Details on test conditions:
TEST SYSTEM
- Ylang Ylang III: WAFs at 0.16, 0.80, 4.0, 20 and 100 mg/L. In addition, 4% and 20% of the WAF at 0.16 mg/L. These latter two concentrations are further indicated as WAFs at 0.0064 and 0.032 mg/L
- Controls: Test medium without test item or other additives.
- Replicates
- 3 replicates of each WAF
- 6 replicates of the control
- 2 extra replicates of the control and WAFs at 0.80 mg/L and higher without algae and without HEPES for TOC analysis
- 1 extra replicate of each test group without algae as a possible background for treated solutions
- Test duration: 72 hours
- Test type: Static
- Test vessels: 40 mL, all-glass, closed with no headspace
- Medium: Adjusted M2
- Cell density: An initial cell density of 1 x 10^4 cells/mL.

OTHER TEST CONDITIONS
- Illumination: Continuously using TLD-lamps with a light intensity within the range of 84 to 85 µE.m-2.s-1.
- Incubation: Airtight closed vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

MEASUREMENT AND RECORDINGS
- pH: At the beginning and at the end of the test.
- Temperature of medium: Continuously in a temperature control vessel.
- Appearance of the cells: At the end of the final test microscopic observations were performed on the WAF at 20 mg /L and the control to observe for any abnormal appearance of the algae.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- 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 680 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

RANGE- FINDING TEST:
A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Three replicates of exponentially growing algal cultures were exposed to WAFs prepared at 0.10, 1.0, 10 and 100 mg/L and to a control
• Cell densities were recorded only at the end of the exposure period
• pH was only measured in the control and the WAF at 100 mg/L
• At the end of the test algae were not observed to verify a normal and healthy appearance

- Results used to determine the conditions for the definitive study: Yes. The expected EL50 for growth rate inhibition was just above a WAF prepared at a loading rate of 100 mg/L. The expected EL50 for yield inhibition was approximating a WAF prepared at a loading rate of 10 mg/L.
Reference substance (positive control):
yes
Remarks:
K2Cr2O7
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:
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
0.73 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.47 - 1.1
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
< 0.006 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on statistical significance,
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
0.032 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on biological significance
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
1.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 1.3 - 2.7
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
< 0.006 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
< 0.006 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
Measured Test Item Concentrations:
TOC concentrations could only be measured in the WAF prepared at 100 mg/L which, was in line with the range finding test. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared.

Inhibition of Growth Rate and Inhibition of Yield:
A flat dose response curve for the inhibition of growth rate was obtained with the loading rates of Ylang Ylang III resulting in 39% inhibition at the highest loading rate of 100 mg/L. Statistically significant inhibition of growth rate was found at all loading rates tested. However, only the inhibition found at loading rates of 0.16 mg/L and higher was found to be biologically relevant, i.e. was >10%. A flat dose response curve for the inhibition of yield was obtained with the loading rates of Ylang Ylang III resulting in 87% inhibition at the highest loading rate of 100 mg/L. Statistically significant inhibition of yield was found at all loading rates tested. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the WAF at 20 mg/L when compared to the control.

Acceptability of the Test:
1. In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 166).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 29%).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 2%).
Results with reference substance (positive control):
Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher.

The EC50 for growth rate inhibition (72h-ERC50) was 0.86 mg/L with a 95% confidence interval ranging from 0.84 to 0.88 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (72h-EYC50) was 0.34 mg/L with a 95% confidence interval ranging from 0.34 to 0.35 mg/L. The historical ranges for yield inhibition lie between 0.20 and 1.1 mg/L. The observed 72h-EYC50 for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
For determination of the NOELR and the EL50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the loading rates compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller). Additionally, the EL10 and EL20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.

Calculation of ELx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding loading rates of the test item. The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Growth Rate (µ, day-1) and Percentage Inhibition for the Total Test Period

Ylang Ylang III,

loading rate (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.702

0.0335

6

0.0064

1.654

0.0463

3

2.8#

0.032

1.602

0.0199

3

5.9#

0.16

1.518

0.0201

3

10.8*

0.80

1.530

0.0383

3

10.1*

4.0

1.448

0.0085

3

14.9*

20

1.458

0.0731

3

14.3*

100

1.034

0.0135

3

39.3*

* Effect was statistically significant

#Effect was statistically significant but biologically not relevant (<10%)

Yield (x104Cells/mL) and Percentage Inhibition for the Total Test Period

Ylang Ylang III,

loading rate (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

164.7

17.20

6

0.0064

142.7

20.12

3

13.4*

0.032

121.2

7.38

3

26.4*

0.16

94.2

5.83

3

42.8*

0.80

97.8

11.45

3

40.6*

4.0

76.1

1.98

3

53.8*

20

79.7

18.46

3

51.7*

100

21.2

0.91

3

87.1*

* Effect was statistically significant

Validity criteria fulfilled:
yes
Remarks:
see details on results
Conclusions:
In conclusion, under the conditions of this study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata),Ylang Ylang III reduced growth rate of this fresh water algae species significantly at a loading rate of 0.16 mg/L and higher (based on biological relevance). The 72h -NOELR, ERL10 and ERL50 was <0.0064 mg/L (when based on statistical analysis and 0.032 mg/L when based on biological relevance), 0.73 and >100 mg/L respectively.

Executive summary:

An OECDTG 201 GLP test was performed with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). Water Accommodated Fractions (WAFs) were prepared and used as test concentrations. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rate of  0.16, 0.80, 4.0, 20 and 100 mg Ylang Ylang III per litre under static conditions. In addition, two lower concentrations were prepared by adding 4% and 20% of the WAF at 0.16 mg/L, respectively, to the test medium.  The initial algal cell density was 10^4 cells/mL and the total exposure period was 72 hours. Samples for Total Organic Carbon (TOC) analyses were taken from WAFs at 0.80 mg/L and higher at the start and at the end of the test. Due to the potential volatile nature of the test item, the exposure was performed in airtight closed vessels with headspace reduced to a minimum.TOC-analysis is a non-specific method and the test substance is a UVCB, therefore the effect parameters were reported in terms of loading rates initially prepared.A flat dose response curve for the inhibition of growth rate was obtained with the loading rates of Ylang Ylang III resulting in 39% inhibition at the highest loading rate of 100 mg/L. Statistically significant inhibition of growth rate was found at all loading rates tested. However, only the inhibition found at loading rates of 0.16 mg/L and higher was found to be biologically relevant, i.e. was >10%.A flat dose response curve for the inhibition of yield was obtained with the loading rates of Ylang Ylang III resulting in 87% inhibition at the highest loading rate of 100 mg/L. Statistically significant inhibition of yield was found at all loading rates tested. The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, under the conditions of this study with Raphidocelis subcapitata, the EL50 of Ylang Ylang III for growth rate inhibition (72h-ERL50) and yield inhibition (72h-EYL50) was >100 mg/L and 1.9 mg/L. While the EL10 for growth rate inhibition (72h-ERL10) and yield inhibition (72h-EYL10) was 0.73 mg/L and <0.0064 mg/L, respectively.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The result derived from read across is sufficiently reliable because all Annex XI criteria are met.
Justification for type of information:
The full read across justification report is attached under "Attached justification".
04 May 2018 READ-ACROSS STUDY - YY EXT/I/II - ALGAL TOX I&B9W8768R001F1.0


Executive Summary
According to Annex VII, 9.1.2 of the REACH Regulation (EC) No 1907/2006, “Growth inhibition study aquatic plants (algae preferred)” (called “algal toxicity in this report) is the standard information required for the registration of substances manufactured or imported in quantities of ten tonnes per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).

An Algae 72-hour toxicity study, according to OECD test guideline 201, is available for the substance Ylang Ylang III oil, which composition is similar to the target substance, with some variations mostly in the concentrations of the constituents.
The compositions of these source and target UVCBs are very close to one another, because the raw material and the distillation process are similar.
All constituents have a sesquiterpene chemical structure, and are thus expected to have similar toxicological properties, essentially though a non-polar narcosis mode of action.
This prediction intends to provide algal toxicity information to assess the classification for aquatic environmental hazards according to CLP Regulation EC/1272/2008.

This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the chemical compositions of both UVCB substances, and the absence of significant difference between the concentrations of the constituents regarding the considered endpoint.
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrates the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, which are summarised .
Reason / purpose:
read-across source
Water media type:
freshwater
Total exposure duration:
72 h
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:
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
0.73 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.47 - 1.1
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
< 0.006 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on statistical significance,
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
0.032 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on biological significance
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
1.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 1.3 - 2.7
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
< 0.006 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
< 0.006 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
Measured Test Item Concentrations:
TOC concentrations could only be measured in the WAF prepared at 100 mg/L which, was in line with the range finding test. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared.

Inhibition of Growth Rate and Inhibition of Yield:
A flat dose response curve for the inhibition of growth rate was obtained with the loading rates of Ylang Ylang III resulting in 39% inhibition at the highest loading rate of 100 mg/L. Statistically significant inhibition of growth rate was found at all loading rates tested. However, only the inhibition found at loading rates of 0.16 mg/L and higher was found to be biologically relevant, i.e. was >10%. A flat dose response curve for the inhibition of yield was obtained with the loading rates of Ylang Ylang III resulting in 87% inhibition at the highest loading rate of 100 mg/L. Statistically significant inhibition of yield was found at all loading rates tested. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the WAF at 20 mg/L when compared to the control.

Acceptability of the Test:
1. In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 166).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 29%).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 2%).
Conclusions:
In conclusion, based on the algae toxicity study for the read across substance Ylang Ylang III oil, the 72h -NOELR, ERL10 and ERL50 for Ylang Ylang I&II was <0.0064 mg/L (when based on statistical analysis and 0.032 mg/L when based on biological relevance), 0.73 and >100 mg/L respectively.



Executive summary:

Toxic effects of Ylang Ylang Ext/I/II to algae is read across from the results of an experimental study with Ylang Ylang III, where Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) was investigated in accordance to GLP and OECD 201 guidelines. The test was performed with Water Accommodated Fractions (WAFs) which were prepared and used as test concentrations. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rate of  0.16, 0.80, 4.0, 20 and 100 mg Ylang Ylang III per litre under static conditions. In addition, two lower concentrations were prepared by adding 4% and 20% of the WAF at 0.16 mg/L, respectively, to the test medium.  The initial algal cell density was 10^4 cells/mL and the total exposure period was 72 hours. Samples for Total Organic Carbon (TOC) analyses were taken from WAFs at 0.80 mg/L and higher at the start and at the end of the test. Due to the potential volatile nature of the test item, the exposure was performed in airtight closed vessels with headspace reduced to a minimum.TOC-analysis is a non-specific method and the test substance is a UVCB, therefore the effect parameters were reported in terms of loading rates initially prepared.A flat dose response curve for the inhibition of growth rate was obtained with the loading rates of Ylang Ylang III resulting in 39% inhibition at the highest loading rate of 100 mg/L. Statistically significant inhibition of growth rate was found at all loading rates tested. However, only the inhibition found at loading rates of 0.16 mg/L and higher was found to be biologically relevant, i.e. was >10%.A flat dose response curve for the inhibition of yield was obtained with the loading rates of Ylang Ylang III resulting in 87% inhibition at the highest loading rate of 100 mg/L. Statistically significant inhibition of yield was found at all loading rates tested. The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, under the conditions of this study with Raphidocelis subcapitata, the EL50 of Ylang Ylang III for growth rate inhibition (72h-ERL50) and yield inhibition (72h-EYL50) was >100 mg/L and 1.9 mg/L. While the EL10 for growth rate inhibition (72h-ERL10) and yield inhibition (72h-EYL10) was 0.73 mg/L and <0.0064 mg/L, respectively.

Description of key information

Toxic effects of Ylang Ylang Ext/I/II to algae is read across from the results of an experimental study with Ylang Ylang III, where Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) was investigated in accordance to GLP and OECD 201 guidelines. The test was performed with Water Accommodated Fractions (WAFs) which were prepared and used as test concentrations. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rate of  0.16, 0.80, 4.0, 20 and 100 mg Ylang Ylang III per litre under static conditions. In addition, two lower concentrations were prepared by adding 4% and 20% of the WAF at 0.16 mg/L, respectively, to the test medium.  The initial algal cell density was 10^4 cells/mL and the total exposure period was 72 hours. Samples for Total Organic Carbon (TOC) analyses were taken from WAFs at 0.80 mg/L and higher at the start and at the end of the test. Due to the potential volatile nature of the test item, the exposure was performed in airtight closed vessels with headspace reduced to a minimum.TOC-analysis is a non-specific method and the test substance is a UVCB, therefore the effect parameters were reported in terms of loading rates initially prepared.A flat dose response curve for the inhibition of growth rate was obtained with the loading rates of Ylang Ylang III resulting in 39% inhibition at the highest loading rate of 100 mg/L. Statistically significant inhibition of growth rate was found at all loading rates tested. However, only the inhibition found at loading rates of 0.16 mg/L and higher was found to be biologically relevant, i.e. was >10%.A flat dose response curve for the inhibition of yield was obtained with the loading rates of Ylang Ylang III resulting in 87% inhibition at the highest loading rate of 100 mg/L. Statistically significant inhibition of yield was found at all loading rates tested. The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, under the conditions of this study withRaphidocelis subcapitata, the EL50 of Ylang Ylang III for growth rate inhibition (72h-ERL50) and yield inhibition (72h-EYL50) was >100 mg/L and 1.9 mg/L. While the EL10 for growth rate inhibition (72h-ERL10) and yield inhibition (72h-EYL10) was 0.73 mg/L and <0.0064 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
0.73 mg/L

Additional information

Values are based on Loading rate (ErLx) and the ErL50 result is a '>'value.

An additional study is available to predict the ErL50 for algae by means of QSAR (Kreatis, 2017). In this study the algae 72h-ErL50 was concluded to be 2.9 mg/L. The experimental value by read across is preferred over the predicted value, as the study was fully valid, the read across substance considered equal or even slightly worst case and resulted in both an ErL50 and concrete ErL10 value, the QSAR only resulted in an ErL50.