Fir, Abies balsamea, ext.

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:

28 May 2018 - 30 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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

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 water (ISO medium, according to OECD 202) was used as dilution water

Details on test solutions:

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 (ISO 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 performed.

Since the stability data of the test item was not available, the test was performed under semi-static conditions. The frequency of the water renewal periods was 24 hours. Prior to treatment of each renewal period, test item solutions were prepared by the method described above.

The test solution was prepared just before introduction of the Daphnia (start of the treatments).

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species and strain: Daphnia magna
Source: In-house laboratory culture with known history
Breeding: The Daphnia are bred in the Ecotoxicological Laboratory of the CRO. The health of the stock animals is continuously monitored by visual daily checking. Abnormal behaviour or significant decrease of population is recorded.
Justification of strain: Daphnia magna is the standard species of the acute immobilisation test.
Number of animals: There were 20 animals in the test and control groups respectively, divided into 4 replicates (5 animals / replicate)
Age of the animals: They were less than 24 h old at the beginning of the test
Acclimatization: There was no acclimatization because the water used was similar to the culture water.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

The test was performed under semi-static conditions. The frequency of the water renewal periods was 24 hours.

Test conditions

Hardness:

The reconstituted water (ISO medium) had a total hardness of 246 mg/L (as CaCO3).

Test temperature:

The water temperature was measured at the start and at the end of the renewal periods in each test vessel. The test temperature was in the range of 20.6 – 20.9°C measured in the test vessels.
The additionally measured temperature in the climate chamber was between 20.1 – 21.0°C

pH:

The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was measured at the start and at the end of the renewal periods in each test vessel and was in the range of 7.69 – 7.94.

Dissolved oxygen:

The dissolved oxygen concentration was measured in each test vessel at the start and at the end of the renewal periods and was in the range of 7.7 – 8.4 mg/L.

Salinity:

not reported

Conductivity:

not reported

Details on test conditions:

Test duration: 48 hours
Test type: Semi-static (Stability of the test item was not available)
Test vessels: 50 mL glass beakers
Medium: Reconstituted water (ISO medium)
Number of daphnids: 20 animals per test group and control group
Loading: 5 per vessel containing 50 mL of test solution
Light: The test was carried out in 16-hour light and 8-hour dark cycle
Feeding: The animals were not fed during the test.

The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.
The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours.
The water temperature, the oxygen concentrations and pH of the control and the test solution were measured at the beginning and at the end of the renewal periods.

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

The date of the last study with reference item Potassium dichromate (batch no.: A0345704) is: 06-07 December 2017.
The 24h EC50: 0.65 mg/L, (95 % confidence limits: 0.61 – 0.70 mg/L)

Any other information on results incl. tables

Validity

 

There were no immobilized animal in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

 

Immobilisation

 

The number of immobilised animals and the percentage of immobility were determined at the 24^thand 48^thhour. In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

 

Table 2:Number and percentage of immobilised animals

Test Group	Number of treated animals	Number of immobilised animals
		24 hours		48 hours
		number	percent	number	percent
Control	20	0	0	0	0
100 %saturated solution (100 mg/Lnominal loading rate)	20	1	5	2	10

 

Although 10 % immobilization could be observed at the test item treated group, this difference was not statistically significant (2 Sample t-Test (a= 0.05) by TOXSTAT software) compared to the control and is negligible due to that the accepted control immobilization is 10 %.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) was assessed with Acute immobilisation test on Daphnia magna, over an exposure period of 48 hours in a semi-static system.

All validity criteria were met during this study.

Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) were the followings:

The 24h and 48h EC50 value: > 100 mg/L nominal loading rate
The 48h EC100 value: > 100 mg/L nominal loading rate
The 48h No-Observed Effect Concentration (NOEC): 100 mg/L nominal loading rate
The 48h Lowest Observed Effect Concentration (LOEC): > 100 mg/L nominal loading rate

Executive summary:

The acute toxicity of Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) on Daphnia magna was assessed in an acute immobilisation test, over an exposure period of 48 hours in a semi-static test system.

Because no toxic response was observed during the preliminary 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 in the definitive test.

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 renewal periods. Measured concentration was below of the Limit of Quantification (LOQ =5 mg Carbon/ L) for all samples during the experiment.

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

Twenty animals, divided into four groups (glass beaker) of five animals each were used at the test concentration and for the control.

All validity criteria were met during this study.

 

Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of Fir Needle Oil Canadian (Fir, Abies Balsamea, ext) were the followings:

The 24h and 48h EC₅₀value:  >100 mg/L nominal loading rate

The 48h EC₁₀₀value: >100 mg/L nominal loading rate

The 48h No-Observed Effect Concentration (NOEC): 100 mg/L nominal loading rate

The 48h Lowest Observed Effect Concentration (LOEC): >100 mg/L nominal loading rate