Storax (balsam)

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

Study period:

29 Oct 2017 - 22 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

Adopted April 13, 2004

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.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Storax (balsam)

EC Number:

232-458-4

EC Name:

Storax (balsam)

Cas Number:

8046-19-3

Molecular formula:

Not applicable, UVCB - NCS

IUPAC Name:

Storax (balsam)

Test material form:

other: liquid

Details on test material:

CAS : 8046-19-3 EINECS : 232-458-4
- Name of test material (as cited in study report): STYRAX RESINOID OIL
- Test Facility test item number: 208399/A
- Appearance: Orange viscous liquid
- Substance type: pure substance
- Physical state: liquid
- Analytical purity: 100%

Specific details on test material used for the study:

Test item information (combined limit range-finding test)
Identification: Styrax resinoid oil
Appearance: Orange viscous liquid
Batch: K17 031-1
Purity/Composition: 100%
Test item storage: At room temperature
Stable under storage conditions until: 02 February 2019
Test Facility test item number: 208399/A

Test item information (final test)
Identification: Styrax resinoid oil
Appearance: Orange viscous liquid
Batch: 0317/1
Purity/Composition: 100%
Test item storage: At room temperature
Stable under storage conditions until: 15 March 2019 (expiry date)
Test Facility test item number: 208399/B
Purity/Composition correction factor: No correction factor required
Chemical name (IUPAC, synonym or trade name: Resinoid obtained from the gum of Liquidambar styraciflua by extraction with solvent
CAS number: 8046-19-3
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Solubility in water: Insoluble
Stability in water: Not indicated

Sampling and analysis

Analytical monitoring:

yes

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: 3.0 mL from the approximate centre of the test vessels
Storage: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling

At the end of the exposure period, the replicates were pooled at each concentration before sampling

Test solutions

Vehicle:

no

Details on test solutions:

Combined limit/range-finding test:
Preparation of test solutions started with loading rates individually prepared at 1.0, 10 and 100 mg/L. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle for 39 minutes. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure. Based on signs of oversaturation at the end of the combined limit/range-finding test, it was decided to adjust the protocol for the preparation of test solutions in the final test.

Final test:
Preparation of test solutions started with loading rates individually prepared at 1.0 to 100 mg/L. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium, whereby stirring speed was reduced compared to the preceding test so that no vortex was visible to minimise the possible formation of an emulsion. The obtained mixtures were allowed to settle for 130 minutes. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure. Observations with a laser pen revealed no Tyndall effect in any of the test solutions. Any residual volumes were discarded.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system:
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 with an age of < 24 hours were selected 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 22 and 23°C during the test.

pH:

pH t=0h : 8.0-8.1
pH t=48h: 7.9- 8.0

Dissolved oxygen:

O2 t=0h : 9.0-9.4
O2 t=48h: 8.3 - 8.8

Nominal and measured concentrations:

Nominal concentrations: WAFs prepared at loading rates of 1.0, 2.2, 4.6, 10, 22, 46 and 100 mg/L.

TOC measurements:
WAFs: t=0h: 0.013, 0.281, 0.888, 0.777, 1.75, 3.69 and 7.64 mg/L
WAFs: t=48h 0.0070, 0.0075, 0.0073, 0.0065, 0.394, 2.73 and 8.01 mg/L

Details on test conditions:

Test Concentrations:
Test item: WAFs prepared at loading rates of 1.0, 2.2, 4.6, 10, 22, 46 and 100 mg/L.
Controls: Test medium without test item or other additives.

Test Procedure and Conditions:
Test duration: 48 hours
Test type: Static
Test vessels: 60 mL, all-glass
Medium: Adjusted ISO medium
Number of daphnids: 20 per concentration
Loading: 5 per vessel containing 50 mL of test solution
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnids: Within 41 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 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 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 loading rates 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.

- Results used to determine the conditions for the definitive study: yes
Considering that at the highest test concentration during the combined limit/range-finding test, daphnids were observed being stuck to each other and that precipitate was present at the end of the test, it seems likely that the highest solution was oversaturated. This assumption is supported by the measured concentration being almost twofold the determined solubility capacity in water at the same loading rate. It was consequently decided to adjust the preparation of test solutions. It was decided to lower the stirring speed to a level that the aqueous phase was still in motion but no vortex was formed to avoid the formation of an emulsion. Additionally the settlement period before the collection of the individual WAFs was extended to ensure optimal phase separation.

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 7.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 7.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Measured concentrations:
Samples taken from all test concentrations were analysed. The concentrations measured at the start of the test were 0.013, 0.28, 0.89, 0.78, 1.8, 3.7 and 7.6 mg/L in WAFs prepared at 1.0, 2.2, 4.6, 10, 22, 46 and 100 mg/L, respectively. These concentrations were at 0.82-105% at the end of the test, whereby the concentrations determined for the lower WAFs decreased more strongly than for the highest two loading rates. Based on these results, the average exposure concentrations were calculated and used to determine the effect parameters.

Immobility:
No immobility was observed in the control or any of the test concentrations, except for one and two daphnids exposed to 0.046 and 7.8 mg/L at the end of the test, respectively. The measured concentrations at the start of the test were in the range of the maximum water solubility of the test item at a loading rate of 100 mg/L. Furthermore, no undissolved material or daphnids sticking to each other were observed at any of the concentrations tested. This indicates that the adjusted protocol for the preparation of test solutions provided suitable solutions for the exposure test without signs of oversaturation. The difference to the results obtained in the preceding combined limit/range-finding test is likely related to the fact that no oversaturation was indicated during the final test and no daphnids got stuck to each other, thus indirectly causing immobility.

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):

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 0.59 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:

No EC50 could be calculated because the test item proved to be non-toxic (EC50 > maximum soluble concentration tested).

Any other information on results incl. tables

Number of Introduced Daphnids and Incidence of Immobility in the Final Test

Time (h)	Replicate	Styrax resinoid oil; Average conc. (mg/L)
		Control	0.0096	0.046	0.080	0.071	0.83	3.2	7.8
0	A	5	5	5	5	5	5	5	5
	B	5	5	5	5	5	5	5	5
	C	5	5	5	5	5	5	5	5
	D	5	5	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20	20	20
24	A	0	0	0	0	0	0	0	0
	B	0	0	0	0	0	0	0	0
	C	0	0	0	0	0	0	0	0
	D	0	0	0	0	0	0	0	0
	Total immobilised	0	0	0	0	0	0	0	0
	Effect %	0	0	0	0	0	0	0	0
48	A	0	0	0	0	0	0	0	0
	B	0	0	0	0	0	0	0	0
	C	0	0	0	0	0	0	0	2
	D	0	0	1	0	0	0	0	0
	Total immobilised	0	0	1	0	0	0	0	2
	Effect %	0	0	5	0	0	0	0	10

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see details on results

Conclusions:

In conclusion, the 48h-EC50 for Daphnia magna exposed to Styrax resinoid oil was beyond the range tested and exceeded a geometric mean concentration of 7.8 mg/L, being considered to be the maximum soluble concentration of the test item in test medium at the highest tested loading level rate (100 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.Water Accommodated Fractions (WAFs) of Styrax resinoid oil were individually prepared at loading rates of 1.0 to 100 mg/L and used as test concentrations.Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAFs prepared at loading rates of 1.0, 2.2, 4.6, 10, 22, 46 and 100 mg/L under static conditions. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test. Samples taken from all test concentrations were analysed. The average (geometric mean) exposure concentrations were calculated and used to determine the effect parameters. No immobility was observed in the control or any of the concentrations tested, except for one and two daphnids exposed to 0.046 and 7.8 mg/L at the end of the test, respectively.The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 48h-EC50 for Daphnia magna exposed to Styrax resinoid oil was beyond the range tested and exceeded a geometric mean concentration of 7.8 mg/L, being considered to be the maximum soluble concentration of the test item in test medium at the highest tested loading level rate (100 mg/L).