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Ecotoxicological information

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:
08 Dec 2017 - 15 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 13, 2004
Qualifier:
according to guideline
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
Identification: Gurjun Balsam Oil (Gurjunene)
Appearance: Pale yellow oily liquid
Batch: 2642704
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 28 February 2018 (expiry date)

Additional information
Test Facility test item number: 209144/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: Essential oil obtained from the resin tapped from Dipterocarpus trees (Dipterocarpaceae) by steam distillation (Gurjunene quality)
Specific gravity / density: 0.9120-0.9330 (20°C)


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
Remarks:
Water Accomodated Fractions (WAFs) were used
Details on test solutions:
The batch of Gurjun Balsam Oil (Gurjunene) tested was a pale yellow oily 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 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
Test temperature:
The temperature continuously measured in a temperature control vessel remained constant at 20°C during the test
pH:
pH t=0h : 7.7- 7.9
pH t=48h: 8.1-8.3



Dissolved oxygen:
O2 t=0h: 8.9- 9.2
O2 t=48h: 8.3 - 8.6

Nominal and measured concentrations:
Nominal concentrations:
WAFs at 0.10, 0.22, 0.46, 1.0, 2.2, 4.6 and 10 mg/L

TOC measurements corrected for for the control treatment
Loading rates: control, 0.10, 0.22, 0.46, 1.0, 2.2, 4.6 and 10 mg/L
WAFs at t=0h: n.a, n.q, n.q, n.q, n.q, 0.44, 0.72 and 1.3 mg TOC/L
WAFs at t=48h n.a, n.q, n.q, n.q, n.q, n.q, 0.43 and 0.82 mg 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 35 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.

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:
• Ten daphnids per concentration (two replicates, five daphnids per replicate) were exposed to WAFs prepared at 0.46, 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.

- Results used to determine the conditions for the definitive study: yes. After 48 hours of exposure ≥60% of the daphnids exposed to WAFs at 1.0 mg/L and higher were immobilized No immobility was observed in the WAF at 0.46 mg/L and the control during the test period. The expected EL50 was between concentrations obtained in WAFs at 0.46 and 1.0 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: 2.0-2.9 mg/L
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% Cl: 4.4-7.2 mg/L
Details on results:
TOC concentrations in the test solutions proved to be too low to measure reliably (almost all <1 mg/L), but were generally in line with the results from the range finding test. After 48 hours of exposure all daphnids exposed to the WAFs at 4.6 and 10 mg/L were immobilized. 30% of the daphnids exposed to the WAF at 2.2 mg/L were immobilized and no immobility was observed in the WAFs at or below 1.0 mg/L and the control during the test period. 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).

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 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 0.87 mg/L with a 95% confidence interval between 0.80 and 0.95 mg/L.
The 48h-EC50 was 0.54 mg/L with a 95% confidence interval between 0.49 and 0.61 mg/L.

Reported statistics and error estimates:
The 24 and 48h-EL50-value were calculated using the Spearman-Karber method (without trim) on the percentages of affected daphnids and the logarithms of thecorresponding loading rates of the test item. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Incidence of Immobility in the Final Test

Gurjun Balsam oil (Gurjunene), loading rate (mg/L)

Immobility (%)

t=24h

Immobility (%)

t=48h

Control

0

0

0.10

5

5

0.22

0

0

0.46

0

0

1.0

0

0

2.2

0

30

4.6

60

100

10

60

100

Validity criteria fulfilled:
yes
Remarks:
see details on results
Conclusions:
The 48h-EL50 for Daphnia magna exposed to Gurjun Balsam Oil (Gurjunene) was 2.4 mg/L based on loading rates (95% confidence interval between 2.0 and 2.9 mg/L).
Executive summary:

An OECDTG 202 GLP test was performed based on the results of a preceding range-finding test. Water Accommodated Fractions (WAFs) of Gurjun Balsam Oil (Gurjunene) were individually prepared at loading rates of 0.10, 0.22, 0.46, 1.0, 2.2, 4.6 and 10 mg/L and used as test concentrations. Twenty daphnids per group (four replicates, five daphnids per replicate) were exposed to a control and to each WAF. 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. 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. TOC in the final test increased with loading in the three highest loadings, indicating proper WAF preparation procedure, and were below detection limit at lower loadings.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 Gurjun Balsam Oil (Gurjunene) was 2.4 mg/L based on loading rates (95% confidence interval between 2.0 and 2.9 mg/L).

 

Description of key information

An OECDTG 202 GLP test was performed based on the results of a preceding range-finding test. Water Accommodated Fractions (WAFs) of Gurjun Balsam Oil (Gurjunene) were individually prepared at loading rates of 0.10, 0.22, 0.46, 1.0, 2.2, 4.6 and 10 mg/L and used as test concentrations. Twenty daphnids per group (four replicates, five daphnids per replicate) were exposed to a control and to each WAF. 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. 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. TOC in the final test increased with loading in the three highest loadings, indicating proper WAF preparation procedure, and were below detection limit at lower loadings.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 Gurjun Balsam Oil (Gurjunene) was 2.4 mg/L based on loading rates (95% confidence interval between 2.0 and 2.9 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
2.4 mg/L

Additional information