Amyris balsamifera, 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

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 analysis

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=48 h
Volume 50 mL
Storage Samples were stored in a refrigerator (2 - 8°C) until analysis.
At the end of the exposure period, the replicates were not pooled at each concentration before sampling. Instead samples were taken from one vessel of each group.
Additionally, reserve samples of 50 mL were taken for possible analysis. If not 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 0.46 and 22 mg/L in the final test. 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.
Any residual volumes were discarded.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source In-house laboratory culture with a known history.
Reason for selection This system has been selected as an internationally accepted invertebrate species.
Validity of batch Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics For the test, young daphnids ware selected with an age of < 24 hours from parental daphnids older than two weeks.

Breeding
Start of each batch: 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.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg/L expressed as CaCO3

Test temperature:

The temperature continuously measured in a temperature control vessel varied between 20 and 21°C during the test

pH:

pH t=0h : 7.8-8.1
pH t=48h: 7.8-7.9

Dissolved oxygen:

O2 t=0h: 8.5-9.0 mg/L
O2 t=48h: 7.3-7.7 mg/L

Nominal and measured concentrations:

Nominal: 0.46, 1.0, 2.2, 4.6, 10, 22 mg/L
TOC measurements corrected for controls:
WAFs: t=0h: 0.59, 0.96, 1.6, 2.9, 8.6, 15 mg/L
WAFs: t=48h -0.12, 0.46, 0.96, 2.8, 8.4, 14 mg/L

TOC measurement test substance: 88.5% TOC

Details on test conditions:

Test duration: 48 hours
Test type: Static
Test vessels: 60 mL, all-glass, airtight closed with headspace reduced to minimum
Medium: Adjusted ISO medium
Number of daphnids: 20 per concentration
Loading: 5 per vessel containing 60 mL of test solution
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnids: Within 32 minutes after preparation of the test solutions.

Immobility (including mortality) was examined at 24 hours and at 48 hours.
pH and dissolved oxygen were measured at the beginning and at the end of the test, for all concentrations and the control.
Temperature of medium were measured continuously in a temperature control vessel, beginning at the start of the test.

The project started with a combined limit/range-finding test. Twenty daphnids per concentration (four replicates, 5 daphnids per vessel) were exposed to a control and a WAF prepared at t a loading rate of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to WAFs prepared at a loading rate of 1.0 and 10 mg/L in the combined range-finding test.
• Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration.

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

5.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval between 4.4 and 6.2 mg/L

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EL0

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

7.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 6.6-8.8

Details on results:

One daphnid was observed trapped at the surface at t = 24h.

The measured TOC concentrations increased with the loading rate at the start of the test indicating proper preparation of WAFs. At the highest concentration 15 mg TOC/L was measured at the start of the test (corresponding to 77% of the total organic carbon expected based on the loading rate was found in the solution). Values below 1.0 mg TOC/L should be considered indicative.

Results with reference substance (positive control):

In conclusion, the actual responses in the reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at Charles River Den Bosch.
The 24h-EC50 was 0.95 mg/L with a 95% confidence interval between 0.85 and 1.1 mg/L.
The 48h-EC50 was 0.59 mg/L with a 95% confidence interval between 0.52 and 0.66 mg/L.

Reported statistics and error estimates:

The 24 and 48h-EL50-value was calculated from the weibits (Weibull analysis) of the percentages of affected daphnids and the logarithms of the corresponding test item loading rates using the maximum likelihood estimation method.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

 

AMYRIS OIL Loading rate (mg/L)	Immobility % t=24h	Immobility % t=48h
Control	0	0
0.46	0	0
1.0	0	0
2.2	0	0
4.6	0	35
10	90	100
22	100	100

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

In the control, no daphnids became immobilised or showed other signs of disease or stress. The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.

Conclusions:

The 48h-EL50 for Daphnia magna exposed to AMYRIS OIL was 5.2 mg/L (95% confidence interval between 4.4 and 6.2 mg/L).

Executive summary:

A full OECDTG 202 GLP test was performed with daphnia magna, based on the results of a preceding range-finding test. Water Accommodated Fractions (WAFs) of Amyris oil were prepared and used as test concentrations.Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAFs individually prepared at loading rates of 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/L under static conditions. The total exposure period was 48 hours and samples for Total Organic Carbon (TOC) analyses were taken at the start and at the end of the test. The exposure was performed in airtight closed vessels with headspace reduced to minimum. 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. In conclusion, the 48h-EL50 for Daphnia magna exposed to AMYRIS OIL was 5.2 mg/L (95% confidence interval between 4.4 and 6.2 mg/L).