Storax (balsam)

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Oct 2017 - 23 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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

corrected 28 July 2011

Test guideline 2

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

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, t=24 h and t=72 h
Volume: 3.0 mL
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 with algae were pooled at each concentration before sampling. Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of Styrax resinoid oil tested was an orange viscous liquid UVCB substance 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.

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 30 minutes. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning through glass wool and used as test concentrations. The highest test concentration was clear and slightly yellow at the end of the preparation procedure, while the lower test concentrations were clear and colorless. 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 133 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. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL. Any residual volumes were discarded.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

ACCLIMATION
- Acclimation period: 3 days
- pre-culturing media and conditions (same as test or not): same

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C

pH:

pH t=0h : 7.6 - 8.1
pH t=48h: 8.3 - 8.6

Nominal and measured concentrations:

Nominal concentrations: WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L

TOC measurements based on 10, 18, 32, 32(without algae), 56 and 100 mg/L
TOC t=0h : 0.021, 0.021, 0.041, 0.041, 0.555, 0.917 mg TOC/L
TOC t=72h: 0.025, 0.023, 0.014, 0.013, 0.052, 0.060 mg TOC/L
TWA: 0.022, 0.021, 0.022, 0.023, 0.097, 0.13 mg/L

Details on test conditions:

TEST CONCENTRATIONS

Test item WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L.
Controls Test medium without test item or other additives.
Replicates - 3 replicates of each test concentration
- 6 replicates of the control
- 1 extra replicate of each test group for sampling purposes after 24 hours of exposure
- 1 or 2 replicates of each test concentration without algae

TEST PROCEDURE AND CONDITIONS
Test duration: 72 hours
Test type: Static
Test vessels: 100 mL, all-glass, containing 50 mL of test solution
Medium: M2
Cell density: An initial cell density of 1 x 10^4 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 85 to 87 μE.m^-2.s^-1.
Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

MEASUREMENTS AND RECORDINGS
-pH: At the beginning and at the end of the test.
-Temperature of medium: Continuously in a temperature control vessel.
-Appearance of the cells: At the end of the final test microscopic observations were performed on all test concentrations and the control to observe for any abnormal appearance of the algae.

EFFECT PARAMETERS MEASURED
After 24 hours of exposure, undissolved particles were observed in the test solutions prepared at loading rates of 32 mg/L and higher, which disturbed spectrophotometric measurement. Therefore algal density was determined by use of a microscope and a counting chamber throughout the test.

TEST CONCENTRATIONS
Spacing factor for test concentrations: 1.8

Combined Limit/Range-Finding Test:
The project started with a combined limit/range-finding test. Six replicates of exponentially growing algae 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:
• Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
• Three replicates per concentration were exposed to WAFs prepared at loading rates of 1.0 and 10 mg/L.
• At the end of the test, algae were observed at only one test concentration and the control to verify a normal and healthy appearance.

Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.13 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.13 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.022 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

based on statistical significance

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 0.13 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

based on biological relevance

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 0.13 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.13 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 0.022 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 8

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.022 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other:

Remarks:

based on statistical significance

Effect concentrations 9

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.022 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other:

Remarks:

based on biological relevance

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.022 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Measured concentrations:
Samples taken from all test concentrations were analysed. The concentrations measured at the start of the test were 0.021, 0.021, 0.042, 0.56 and 0.92 mg/L in WAFs prepared at 10, 18, 32, 56 and 100 mg/L, respectively. These concentrations were at 6.5-118% at the end of the test, whereby the concentrations determined for the lower WAFs decreased less strongly than for the higher loading rates. The concentrations measured in the samples without algae were comparable to the concentrations in the samples with algae. Based on these results, the Time Weighted Average (TWA) exposure concentrations were calculated and used to determine the effect parameters.

Inhibition of Growth Rate and Inhibition of Yield:
Growth rate inhibition increased slightly with increasing test concentration but was overall in a comparable range at all concentrations with a statistically significant difference to the control group. Inhibition was, however, below 10% at all test concentrations and thus considered to be biologically not relevant. The NOEC for growth rate inhibition was set at 0.13 mg/L.
Inhibition of yield increased slightly with increasing concentration of Styrax resinoid oil but was overall in a comparable range (20-29% inhibition) at all concentrations with a statistically significant difference to the control group.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to all concentrations tested when compared to the control. The measured concentrations were below the water solubility of the test item throughout the test. This is likely related to the test medium containing substances for the support of algal growth. After 24 hours of exposure, undissolved particles were observed in solutions prepared at loading rates of 32 mg/L and higher, indicating that the test item or the medium changed composition during the test, which could explain the stronger decrease of test item concentrations during the test at the two highest test levels. No haziness was observed as compared to the preceding test, and the concentrations measured at the start of the test were more representative of the applied range of loading rates. This indicates that the adjusted protocol for the preparation of test solutions provided suitable solutions for the exposure test. The difference to the growth rate results obtained in the preceding combined limit/range-finding test was likely related to the fact that no oversaturation of test item was indicated during the final test with a hazy or slightly coloured test solution interacting with the availability of nutrients or light. Furthermore, the algal culture used for the final test grew faster and the validity criteria were met, indicating that the culture used in the combined limit/range-finding test might have been of compromised quality prior to the test.


Acceptability of the Test:
1. In the controls, cell density increased by an average factor of at least 16 within the exposure period (i.e. 128).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 18%).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 4.1%).

Results with reference substance (positive control):

The EC50 for growth rate inhibition (72h-ERC50) was 1.1 mg/L with a 95% confidence interval of 1.1 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range. The EC50 for yield inhibition (72h-EYC50) was 0.36 mg/L with a 95% confidence interval ranging from 0.35 to 0.36 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested was below this range. Nevertheless, this was accepted as the EC50 for growth rate was within historical range

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).

Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. No EC50-values could be calculated because the test item proved to be non-toxic (EC50 > maximum soluble concentration tested). The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Inhibition results

Growth Rate And Percentage Inhibition For The Total Test Period

 

Styrax resinoid oil TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.612	0.0656	6
0.022	1.542	0.0199	3	4.3*^#
0.021	1.531	0.0418	3	5.1*^#
0.022	1.519	0.0199	3	5.8*^#
0.097	1.517	0.0630	3	5.9*^#
0.13	1.502	0.0548	3	6.8*^#

* effect was statistically significant;^#effect biologically not relevant (<10%)

Yield And Percentage Inhibition For The Total Test Period

Styrax resinoid oil TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	127.0	24.19	6
0.022	101.3	6.21	3	20*
0.021	98.2	12.00	3	23*
0.022	94.3	5.77	3	26*
0.097	94.8	17.21	3	25*
0.13	90.5	15.50	3	29*

* effect was statistically significant

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See details on results

Conclusions:

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, no biologically relevant inhibition of growth rate was recorded at any of the concentrations of Styrax resinoid oil tested. The 72h ErL50 for P. subcapitata was beyond the range tested and exceeded a TWA concentration of 0.13 mg/L.

Executive summary:

A full OECDTG 201 GLP test was performed with Pseudokirchneriella subcapitata. A final test was performed based on the results of a preceding combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg Styrax resinoid oil per litre. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.  A slight dose-related increase of inhibition of both growth rate and yield was observed at the end of the test. An inhibition of 6.8% and 29% was observed at the highest concentration tested for growth rate and yield, respectively. Time Weighted Average (TWA) exposure concentrations were calculated and used to determine the effect parameters. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, no biologically relevant inhibition of growth rate was recorded at any of the concentrations of Styrax resinoid oil tested. Both the EC50 and EC10 for growth rate inhibition (72h-ErC50, ErC10) and EC50 for yield inhibition (72h-EyC50) were beyond the range tested and exceeded a TWA concentration of 0.13 mg/L. The 72h-NOEC for growth rate inhibition was 0.13 mg/L based on biological relevance, while the 72h-NOEC for yield inhibition was below 0.022 mg/L.