Fir, Abies balsamea, ext.

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 May 2018 - 31 May 2018

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:

28 July 2011

Deviations:

yes

Remarks:

The differences in light intensity between the test vessels exceeded 15 % variation in one case out of ten. No presumed effect on study

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA Ecological Effects Test Guidelines, OCSPP 850.5400, Algal Toxicity, Tiers I and II, EPA 712-C-96-164

Version / remarks:

April 1996

Deviations:

yes

Remarks:

The differences in light intensity between the test vessels exceeded 15 % variation in one case out of ten. No presumed effect on study

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fir, Abies balsamea, ext.

EC Number:

285-364-0

EC Name:

Fir, Abies balsamea, ext.

Cas Number:

85085-34-3

IUPAC Name:

Fir Balsam Oil

Test material form:

liquid

Details on test material:

Name of test material as cited in study report: Fir Needle Oil Canadian (Fir, Abies Balsamea, ext)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Botanical source and Lot# 67919
- Expiration date of the lot/batch: 13 septembre 2018
- Purity test date: UVCB, considered 100% pure

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25 °C, ≤ 70 RH%), protected from light, avoid contact with iron.

Sampling and analysis

Analytical monitoring:

yes

Remarks:

TOC analysis

Details on sampling:

Analytical measurement was performed by TOC analysis and therefore only the sum of the carbon content of the test item could be measured this way. Samples were taken at the tested concentration level as well as from the control at the beginning and at the end of the renewal periods to justify that the test item is present in the test solution, since more accurate data cannot be provided by TOC.
Samples were taken from the test solution and the control solution. the samples were filled into the tubes and they were measured by the NPOC method without any dilution. The measured carbon contents of the test solutions were corrected with the measured values of the control samples.

Test solutions

Vehicle:

no

Remarks:

Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water

Details on test solutions:

Test item formulation
Because the test item is poorly soluble in water, a test solution was prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
A saturated test item solution (nominal loading rate of 100 mg/L) was prepared by dispersing/dissolving the amount of test item into the test medium (OECD Medium) two days before the start of the study. This solution was shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give the 100 % saturated solution.
As a Limit Test was carried out, further dilution of stock solution was not be performed.

Untreated Control
Algal growth medium (OECD Medium) was inoculated with algal cells (without test item) and was examined in parallel to the test item concentrations.

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of the CRO
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Breeding conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, after an incubation period of three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

not reported

Test temperature:

Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.6 – 22.8 °C measured in the flask and between 22.1 and 23.4 °C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the test in each test vessels. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.80 – 9.06 during the experiment.

Dissolved oxygen:

Measured concentration was below of the Limit of Quantification (LOQ = 5 mg Carbon/ L) both at the start and at the end of the experiment.
The biological results are based on the nominal test item concentration.

Salinity:

not reported

Conductivity:

not reported

Details on test conditions:

Light Intensity
The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 8358 lux (equivalent to ~113 microE/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels were within 15 % (with the exception of only one case where the difference was 16.75 %, see Section 3.12.) and therefore provided equal conditions for each test vessel.

The test was started (0 hours) by inoculation of a biomass of approximately 10000 algal cells per mL test medium.

The test was performed with six replicates per test concentration and six replicates in the control group. Volumes of 100 mL test solution per replicate in 250 mL sterile Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.
All procedures were performed under sterile conditions during the test.

The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satis factory test conditions.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

not specified

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

not specified

Results with reference substance (positive control):

The date of the last study with the reference item Potassium dichromate is (Batch Number: A0345704): 04 – 07 December 2017.
The 72h ErC 50 (biomass): 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EbC 50 (growth rate): 0.63 mg/L, (95 % confidence limits: 0.58 – 0.69 mg/L)
The 72h EyC 50 (yield): 0.53 mg/L, (95 % confidence limits: 0.49 – 0.58 mg/L)

These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Any other information on results incl. tables

Validity

 

The cell density in the control cultures increased by the factor of 73.17 within three days.

The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 11.74 %.

The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.71 %.

All validity criteria were met, therefore the study can be considered as valid.

Average Specific Growth Rates

The results of the statistical evaluation (based on 2 Sample t-Test;a=0.05) show that the 0-72 h average specific growth rate was not statistically significantly different from the untreated control value

Areas under the growth curves

The results of the statistical evaluation (based on 2 Sample t-Test;a=0.05) show that the 0-72 h areas were not statistically significantly different from the untreated control value

Yield

The results of the statistical evaluation (based on 2 Sample t-Test;a=0.05) show that the 0-72 h yield was not statistically significantly different from the untreated control value

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS

There were no any observed morphological deviations during the experiment.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), over an exposure period of 72 hours.

Under the conditions of this algal growth inhibition test the calculated endpoints for the effect of Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) were the following:

The 72h EbC50 value (biomass): > 100 mg/L nominal loading rate
The 72h ErC50 value (growth rate): > 100 mg/L nominal loading rate
The 72h EyC50 value (yield): > 100 mg/L nominal loading rate

The 72h No-Observed Effect Concentration (NOEC): 100 mg/L nominal loading rate
The 72h Lowest Observed Effect Concentration (LOEC): > 100 mg/L nominal loading rate

Executive summary:

The effect of Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours.

Because a toxic response was not observed during the preliminary concentration range-finding test, a Limit Test was carried out using only one test concentration at the solubility level of the test item in the test medium (100 mg/L nominal loading rate, 100 % saturated solution) and one control group.

Analytical measurement was performed by TOC analysis and therefore only the sum of the carbon content of the test item was measured this way. Samples were taken at the tested concentration level as well as from the control at the start and at the end of the experiment. Measured concentration was below of the Limit of Quantification (LOQ =5 mg Carbon/ L) both at the start and at the end of the experiment.

The biological results are based on the nominal test item concentration.

The test design included six replicates at test concentration and six replicates for the untreated control.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (a= 0.05) by TOXSTAT software.

The E_rC_50,E_bC₅₀and E_yC₅₀values of the test item were determined directly from the raw data.

 

Under the conditions of this algal growth inhibition test the calculated endpoints for the effect of Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) were the following:

The 72h E_bC₅₀value (biomass): >100 mg/L nominal loading rate

The 72h E_rC₅₀value (growth rate): >100 mg/L nominal loading rate

The 72h E_yC₅₀value (yield): >100 mg/L nominal loading rate

The72h No-Observed Effect Concentration (NOEC): 100 mg/L nominal loading rate

The72h Lowest Observed Effect Concentration (LOEC): >100 mg/L nominal loading rate