Amyris balsamifera, ext.

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:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, 2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Amyris balsamifera, ext.

EC Number:

291-076-6

EC Name:

Amyris balsamifera, ext.

Cas Number:

90320-49-3

Molecular formula:

Not applicable due to the UVCB nature of the substance.

IUPAC Name:

Essential oil of Amyris obtained from the wood of Amyris balsamifera (Rutaceae) trees by steam distillation.

Test material form:

liquid: viscous

Remarks:

Pale yellow slightly viscous liquid

Details on test material:

Name of test material as cited in study report: Amyris oil
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from light

Specific details on test material used for the study:

Identification: AMYRIS OIL
Appearance: Pale yellow slightly viscous liquid
Batch: L4275185
Purity/Composition: UVCB
Test item storage: At room temperature protected from light
Stable under storage conditions until 31 October 2017 (retest date)

Test item: 207778/A
Purity/composition correction factor: No correction factor required
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Volatile: Not indicated
Solubility in water: 1:1 in ethanol 90%
Stability in water: Not indicated

Sampling and analysis

Analytical monitoring:

yes

Remarks:

TOC measurement

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control 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.
At the end of the exposure period, the replicates with algae were not pooled at each concentration before sampling.
Additionally, reserve samples of 40 mL were taken from all test solutions 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.
No. of repeats: at least 3

Test solutions

Vehicle:

no

Remarks:

Water Accomodated Fractions (WAFs) were used

Details on test solutions:

The batch of AMYRIS OIL tested was a pale yellow slightly viscous liquid. The test item was a UVCB substance not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with loading rates individually prepared ranging between 1.0 and 100 mg/L in the combined limit/range-finding test and between 0.25 and 64 mg/L in the final test. Exact amounts of test item were added to test medium containing no HEPES buffer. A 2-day period of magnetic stirring in closed vessels with minimal headspace and in the dark was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle overnight. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning through glass wool and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure. In addition, microscopic observation of WAFs showed that they did not contain undissolved test material.
For the combined limit/range-finding test, after preparation, volumes of 110 mL were added to each replicate of the respective test concentration. Subsequently, 2.2 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL and HEPES buffer (0.66 mL) was spiked to each vessel. For the final test, after preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL and HEPES buffer (0.24 mL) was spiked to each vessel.

Solutions for analysis of TOC concentrations:
• At the start of the test: samples were taken from freshly prepared solutions (before preparation of the exposure vessels).
• At the end of the exposure period: volumes of 110 mL (combined limit/range-finding test) or 40 mL (full test) of each WAFs were added to vessels with no algae at the start of the test and used as solutions for analysis of TOC concentrations at the end of the test. These vessels were not spiked with HEPES buffer. This was done to prevent that the carbon originating from the buffer will obscure the results of TOC analysis.
Any residual volumes were discarded.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: 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 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).

pH:

t=0h : 7.3 - 7.4
t =72h: 7.3 - 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: 0.25, 1.0, 4.0, 16, 64 mg/L
TOC measurements corrected for controls:
TOC t=0h : -0.23, 0.49, 3.1, 10.0, 25 mg/L
TOC t=72h: -0.41, 0.77, 1.7, 7.2, 23 mg/L

The TOC content of the test item was estimated to be 88.49%

Details on test conditions:

AMYRIS OIL: WAFs prepared at loading rate of 0.25, 1.0, 4.0, 16 and 64 mg/L .
Controls: Test medium without test item or other additives.
Replicates: 3 replicates of each test concentration; 6 replicates of the control; 2 extra replicates of each test concentration and the control without algae for sampling purposes; 1 extra replicate of each test item concentration without algae as a background for treated solutions.

Test duration : 72 hours
Test type: Static
Test vessels: 40 mL, airtight closed with no headspace to prevent any loss of the test item due to volatilization.
Medium: Adjusted M2
Cell density: An initial cell density of 1 x 10^4 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 81 to 89 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.
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

A spacing factor of 4 was used as the dose-response recorded in the combined limit/range-finding test was shallow.

The project started with a combined limit/range-finding test.
Six replicates of exponentially growing algae were exposed to a control and WAF prepared at a loading rate of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Three replicates per concentration were exposed to WAFs prepared at loading rates of 1.0 and 10 mg/L.
• Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
• At the end of the exposure period, pH was only measured in the control and the highest test concentration.
• At the end of the test algae were not observed to verify a normal and healthy appearance.
• 50 mL samples were taken for analysis

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 17-19

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 4.1-5.1

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on biological significance

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95%CL: 2.8-3.7

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 0.13-0.24

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

< 0.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The measured TOC concentrations increased with the loading rate at the start of the test indicating proper preparation of WAFs.

In the control, cell density increased by an average factor of 318.
The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 18%.
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 2.2%.

Results with reference substance (positive control):

The EC50 for growth rate inhibition (72h-ERC50) was 1.2 mg/L with a 95% confidence interval ranging from 1.1 to 1.2 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72h-EYC50) was 0.43 mg/L with a 95% confidence interval ranging from 0.42 to 0.44 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations 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).

Any other information on results incl. tables

Growth Rate (µ, Day^-1) And Percentage Inhibition For The Total Test Period

AMYRIS OIL Loading rate (mg/L)	%Inhibition
Control
0.25	9.4^#
1.0	3.3^#
4.0	6.7^#
16	46*
64	87*

* - effect was statistically significant,^#- effect was statistically significant but biologically irrelevant (<10%)

Yield (x10⁴Cells/mL) And Percentage Inhibition For The Total Test Period

AMYRIS OIL Loading rate (mg/L)	%Inhibition
Control
0.25	42*
1.0	18*
4.0	32*
16	93*
64	100*

* - effect was statistically significant

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see details on results

Conclusions:

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, the EL50 for growth rate inhibition (72h-ERL50) was 18 mg/L with a 95% confidence interval ranging from 17 to 19 mg/L.
The EL50 for yield inhibition (72h-EYL50) was 3.2 mg/L with a 95% confidence interval ranging from 2.8 to 3.7 mg/L.
The 72h-NOEL for growth rate inhibition was 4.0 mg/L based on biological relevance and below 0.25 mg/L based on statistical significance.
The 72h-NOEL for yield inhibition was below 0.25 mg/L.

Executive summary:

A full OECDTG201 GLP test was performed with Pseudokirchneriella subcapitata. Six exponentially growing algal cultures were exposed for 72h to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rate of 0.25, 1.0, 4.0, 16 and 64 mg AMYRIS OIL per liter. The total exposure period was 72 hours and samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of exposure. The measured TOC concentrations increased with the loading rate at the start of the test indicating proper preparation of WAFs. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. Statistically significant inhibition of growth rate was found at all loadings tested. Inhibition of yield was both statistically significant and biologically relevant at all concentrations. The EL50 for growth rate inhibition (72h-ERL50) was 18 mg/L with a 95% confidence interval ranging from 17 to 19 mg/L. The EL10 was 4.6 mg/L (4.1-5.1; 95%CL). The EL50 for yield inhibition (72h-EyL50) was 3.2 mg/L with a 95% confidence interval ranging from 2.8 to 3.7 mg/L. The 72h-NOEL for growth rate inhibition was 4.0 mg/L based on biological relevance and below 0.25 mg/L based on statistical significance. The 72h-NOEL for yield inhibition was below 0.25 mg/L.