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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Jun 2017 - 08 Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 13, 2004
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures
Version / remarks:
OECD series on testing and assessment number 23, 2000.
GLP compliance:
yes
Specific details on test material used for the study:
Test item information, batch used for the combined limit/range-finding test and the range-finding test:
Test Facility test item number: 208293/A
Identification: Ylang Ylang III
Appearance: Pale yellow clear liquid (determined by Charles River Den Bosch)
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 31 August 2018 (expiry date)

Test item information, batch used for the final test:
Test Facility test item number: 208293/B
Identification: Ylang Ylang III
Appearance: Clear pale yellow liquid (determined by Charles River Den Bosch)
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 30 September 2019 (expiry date)

Additional information
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: 83863-303 (8006-81-3)
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Volatile: Not indicated
Solubility in water: Not available (UVCB)
Stability in water: Not indicated
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: 40 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 40 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.

Vehicle:
no
Details on test solutions:
The batches of Ylang Ylang III tested were clear pale yellow liquids. The test items were UVCBs and not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test items. During preparation of test solutions the test items were 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. All test solutions were clear and colorless at the end of the preparation procedure. Any residual volumes were discarded.
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 selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.

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).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
180 mg/L expressed as CaCO3
pH:
pH t=0h : 7.9
pH t=48h: 8.0-8.1
Dissolved oxygen:
O2 t=0h: 8.9 - 9.4
O2 t=48h: 7.8 - 8.6
Nominal and measured concentrations:
Nominal concentrations: WAFs at 0.10, 0.22, 0.46, 1.0, 2.2 and 4.6 mg/L

TOC measurements corrected for controls:
WAFs: t=0h: n.a., n.q, n.q, n.q, n.q, n.q, n.q, mg TOC/L
WAFs: t=48h n.a., n.q, n.q, n.q, 0.29, n.q, 0.3mg TOC/L

n.a. – not applicable, n.q. – not quantifiable
Details on test conditions:
Test system:
Test duration: 48 hours
Test type: Static
Test vessels: 60 mL, all-glass, closed with no headspace
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 25 minutes after preparation of the test solutions.

Measurements and Recordings:
Immobility (including mortality): At 24 hours and at 48 hours.
pH and dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control.
Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

Combined Limit/Range-Finding Test
The study started with a combined limit/range-finding test. Twenty daphnids per concentration (four replicates, five daphnids per replicate) were exposed to a control and a WAF at 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Ten daphnids per concentration (two replicates, five daphnids per replicate) were exposed to WAFs at 1.0 and 10 mg/L;
• Dissolved oxygen concentrations and pH were only measured in the control and the WAF at 100 mg/L.

Range-Finding Test:
An additional 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:
• Ten daphnids per concentration (two replicates, five daphnids per replicate) were exposed to WAFs at 0.10, 1.0, 10 and 100 mg/L and to a control;
• Dissolved oxygen concentrations and pH were only measured in the control and the WAF at 100 mg/L.

Reference substance (positive control):
yes
Remarks:
K2Cr2O7
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
2.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL (0.91 - 5.9 mg/L)
Duration:
48 h
Dose descriptor:
EL0
Effect conc.:
0.46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 4.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
TOC concentrations in the test solutions proved to be too low to measure reliably (all < 1 mg/L), but are in line with results from the range finding tests. After 48 hours of exposure 38, 30 and 85% of the daphnids exposed to the WAFs at 1.0, 2.2 and 4.6 mg/L, respectively were immobilized. No immobility was observed in the WAFs at or below 0.46 mg/L and the control during the test period.These test conditions remained within the limits prescribed by the study plan (pH: 6-9, not varying by more than 1.5 units; oxygen: >= 3 mg/L at the end of the test). The temperature continuously measured in a temperature control vessel was maintained at 20°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C).

Acceptability of the Test:
1. In the control , no daphnids became immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium.
2. The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.
Results with reference substance (positive control):
In conclusion, the actual responses in this reference test with K2Cr2O7 were 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 0.59 mg/L with a 95% confidence interval between 0.52 and 0.68 mg/L.
The 48h-EC50 was 0.41 mg/L with a 95% confidence interval between 0.34 and 0.46 mg/L.
Reported statistics and error estimates:
The 24h-EL50 could not be determined because the observed effects were below 50%. The 48h-EL50 was calculated from the weibits of the percentages of affected daphnids and the logarithms of the corresponding loading rates of test item using the maximum likelihood estimation method. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Incidence of Immobility in the Final Test

Ylang Ylang III, loading rate (mg/L)

Immobility %

t=24h

 

Immobility %

t=48h

Control

0

0

0.10

0

0

0.22

0

0

0.46

5*

0*

1.0

0

38

2.2

0

30

4.6

5

85

  *Immobilisation is not an irreversible effect.

Validity criteria fulfilled:
yes
Remarks:
see details on results
Conclusions:
The 48h-EL50 for Daphnia magna exposed to Ylang Ylang III was 2.4 mg/L based on loading rates (95% confidence interval between 0.91 and 5.9 mg/L).
Executive summary:

A full OECDTG 202 GLP test was performed with Daphnia magna, based on the results of a preceding combined limit/range-finding test test. Water Accommodated Fractions (WAFs) of Ylang Ylang III  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 0.10, 0.22, 0.46, 1.0, 2.2 and 4.6 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 showed that the test item was poorly soluble in test medium and the results were inconclusive. For these reasons and because the TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 48h-EL50 for Daphnia magna exposed to Ylang Ylang III was 2.4 mg/L based on loading rates (95% confidence interval between 0.91 and 5.9 mg/L).

 

Description of key information

A full OECDTG 202 GLP test was performed with Daphnia magna, based on the results of a preceding combined limit/range-finding test test. Water Accommodated Fractions (WAFs) of Ylang Ylang III  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 0.10, 0.22, 0.46, 1.0, 2.2 and 4.6 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 showed that the test item was poorly soluble in test medium and the results were inconclusive. For these reasons and because the TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 48h-EL50 for Daphnia magna exposed to Ylang Ylang III was 2.4 mg/L based on loading rates (95% confidence interval between 0.91 and 5.9 mg/L).

 

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
2.4 mg/L

Additional information

Value is based on loading: EL50