Balsams, gurjun

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:

Balsams, gurjun

EC Number:

232-444-8

EC Name:

Balsams, gurjun

Cas Number:

8030-55-5

IUPAC Name:

Essential oil of Gurjun obtained from the resin tapped from Dipterocarpus trees (Dipterocarpaceae) by steam distillation (Copaene quality)

Test material form:

liquid

Remarks:

Clear colourless to pale yellow liquid

Details on test material:

- Name of test material (as cited in study report): Gurjun Balsam oil
- Substance type: pure substance
- Physical state: liquid
- Analytical purity: no data
- Lot/batch No.: confidential information

Specific details on test material used for the study:

Identification: GURJUN BALSAM OIL
Appearance: Clear colourless to pale yellow liquid
Batch: L4285828
Purity/Composition: UVCB
Test item storage: At room temperature protected from light
Stable under storage conditions until: 30 April 2018 (retest date)

Test item: 207786/A
Purity/composition correction factor: No correction factor required
CAS Number: 8030-55-5 (93165-18-5)
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Volatile: Not indicated
Solubility in water: Insoluble
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 GURJUN BALSAM OIL tested was a clear colourless to pale yellow liquid. The item was a UVCB substance poorly 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 0.10 and 2.2 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 and used as test concentrations. As the two highest concentrations in the combined limit/range-finding test contained undissolved test material when observed under microscope and the highest concentration was hazy, it was decided to use glass wool to siphon test solutions in the final test. All test solutions prepared for the final test 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 were selected with an age of < 24 hours from parental daphnids older than two weeks.

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, and complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C).

pH:

pH t=0h : 8.0 - 8.2
pH t=48h: 8.1 - 8.4

Dissolved oxygen:

O2 t=0h: 9.3 - 9.6 mg/L
O2 t=48h: 7.7 - 8.7 mg/L

Nominal and measured concentrations:

Nominal: 0, 0.10, 0.22, 0.46, 1.0, 2.2 mg TOC/L
TOC measurements corrected for controls:
WAFs: t=0h: n.a., 0.048, 0.087, 0.068, 0.39, 0.35 mg/L
WAFs: t=48h n.a., 0.24, 0.23, 0.22, 0.074, 0.00030 mg/L

TOC measurement test substance: 84.39% 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, however 21 in 0.46 mg/L and 19 in 1 mg/L
Loading: 5 per vessel containing 60 mL of test solution (Final test: 6 instead of 5 daphnids were exposed to WAF of 0.46 mg/L in vessel D, whereas, 4 instead of 5 were exposed to WAF of 1.0 mg/L in vessel A.)
Evaluation: These numbers were taken into account during the calculation of effects.

Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnids: Within 26 minutes after preparation of the test solutions

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.

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.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval between 0.34 and 0.48 mg/L

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EL0

Effect conc.:

0.22 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.:

> 2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: as observed in the full test

Effect concentrations 4

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: as observed in the combined limit/range-finding test

Details on results:

The measured TOC concentrations were below 1.0 mg/L at the start of the test. This value is considered a sensitivity limit of the method, and therefore, obtained values should be considered indicative. The values measured at the start of the test showed slight increase with the applied dose. A concentration of approximately 0.40 mg/L was measured at the two highest concentrations tested indicating a possible solubility limit in test medium.
At the end of the test a significant decrease of the measured TOC concentrations was observed at the two highest loading rates.

No immobility was observed in the control and at the two lowest test concentrations during the entire exposure period. At 24 hour of exposure 11 and 15% of daphnids were immobilized in WAFs of 1.0 and 2.2 mg/L, respectively.
At the end of the test, the observed effects were not increasing with the applied dose; in contrary, immobilization decreased with applied dose after reaching 100% at WAF of 0.46 mg/L. One possible explanation for the observed effects can be the decrease of the concentration in the two highest groups at the end of the test. Nevertheless, the immobilization observed at WAF of 1.0 mg/L was above 50% in both, the combined limit/range-finding test and the final test. Therefore, for the worst-case-scenario the effects parameters were calculated after exclusion of the highest WAF.

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 0.95 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:

Calculation of EL50:
The 24- EL50-value could not be calculated as the effects observed at 24 hour of exposure were below 50%. The 48h-EL50-value was calculated with the Spearman-Karber procedure. The highest WAF was not included in the analysis .

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

 

Gurjam balsam oil Loading rate (mg/L)	Immobility % t=24h	Immobility % t=48h
Control	0	0
0.10	0	0
0.22	0	0
0.46	0	100 (21/21)
1.0	11 (2/19)	68 (13/19)
2.2	15 (3/20)	20 (4/20)

  () between brackets: number of immobilised/number introduced daphnids

Measured TOC concentrations in the final test

GURJUN BALSAM OIL Loading rate (mg/L)	Measured concentrations (mg TOC/L)*		Relative to nominal loading rate (%)
	T=0h	T=48h	T=0h	T=48h
Control	n.a.	n.a.	n.a	n.a.
0.10	0.084	0.24	100	286
0.22	0.087	0.23	47	124
0.46	0.068	0.22	17	57
1.0	0.39	0.074	46	8.8
2.2	0.35	0.00030	19	0.016

* corrected for the control treatment

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 GURJUN BALSAM OIL was 0.40 mg/L (95% confidence interval between 0.34 and 0.48 mg/L).

Executive summary:

A full OECDTG 202 GLP test was performed with daphnia magna, based on the results of a preceding limit/range-finding test. Water Accommodated Fractions (WAFs) of GURJUN BALSAM OIL oil were prepared and used as test concentrations.Twenty daphnids per group (5 per replicate, Quadruplicate) were exposed to an untreated control and to Water Accommodated Fractions (WAFs) individually prepared at loading rates of 0.22, 0.46, 1.0, 2.2 and 4.6 mg/Lunder 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 were below 1.0 mg/L at the start of the test. This value is considered a sensitivity limit of the method, and therefore, obtained values should be considered indicative. The values measured at the start of the test showed a slight increase with the applied dose. A concentration of approximately 0.40 mg/L was measured at the two highest concentrations tested indicating a possible solubility limit in test medium. 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 GURJUN BALSAM OIL was 0.40 mg/L (95% confidence interval between 0.34 and 0.48 mg/L).