Resinoid of Abies balsamea (Pinaceae) obtained from needles by an organic solvent extraction

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-20 to 2015-08-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

referenced as Method C.2 of Commission Regulation No. 440/2008

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Not applicable

Analytical monitoring:

yes

Details on sampling:

Chemical analyses: Single samples for analysis were taken from all test solutions at the start of the test (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

Vehicle:

no

Details on test solutions:

The study was carried out using WAFs (Water Accommodated Fractions) since the test item was not well soluble in test water. The WAFs (for the fresh media at t=0h and t=24h) were prepared under closed conditions and by slow-stirring to avoid production of a dispersion.
The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the WAFs. The volume of the mixing vessel was approximately 1L. A magnetic stirring bar was placed in each mixing vessel completely filled with test water (without headspace). The loadin rates of the test item were weighed in glass flasks (approximate volume: 100 mL) filled with minimum headspace with test water (from the mixing vessel) and were immediately sealed with screwcaps after weighing. Each glass flask was placed in a water bath for 10-15 minutes at 50°C, followed by sonication for 5 to 10 minutes. Based on experience on similar substances, these steps (heating and sonication) were essential to remove the gum pellets stuck to the glass of the flasks (especially at the highest loading rates) and to extract the soluble fraction of the test item as possible. Then the mixing vessels were carefully filled with the contents of the glass flasks and thereafter were closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 +/- 2 hours of gentle stirring at room temperature, the WAFs were allowed to stand for 1 hour before use and were extracted via the drain port. The first 100 mL were discarded. Then the WAFs were directly added into test tubes (without headspace) that were immediately sealed with screwcaps after introduction of daphnids. No small bubble was observed in the test tubes. The test solution was observed to be clear and colourless at all loading rates. The test was carried out without adjustment of the pH.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Strain/clone: clone 5
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Sex: Female
- Age at Test Start: < 24 hours old
- Feeding during test: No feeding
- Validity of Batch: Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Breeding conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the
test. The cultivation of the parental daphnids was performed in all-glass beakers containing purified drinking water (Composition (mg.L-1): Calcium 12 – Magnesium 8.0 – Sodium 12 – Potassium 6 – Silica 32 – Bicarbonates 74 – Sulphates 9 – Chlorides 15 – Nitrates 7.3; pH = 7). Cultures were maintained at a density of 1 adult daphnid per 25 mL of
- Acclimation: At least 48 hours prior to the start of the test, gravid daphnids were transferred to OECD test water and held at similar temperature and light conditions as used in the test. During this period, daphnids were fed in the same manner as that of the stock population. Only daphnids up to 24 hours old were used for the test.

Test type:

semi-static

Water media type:

freshwater

Remarks:

Reconstituted water, as prescribed by the OECD Guideline 202

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Hardness:

250 mg.L-1 (as CaCO3)

Test temperature:

Between 20.6 and 21.3 °C throughout the test (average value: 20.9°C).
This complied with the requirements of the guideline (20°C ± 2°C, constant within 1°C).

pH:

6.86 to 7.78.
Throughout the test, the pH remained within the prescribed values (6.0-9.0, not varying by more than 1.5 units)

Dissolved oxygen:

6.84 to 8.65
Dissolved oxygen concentration at the end of the test was ≥ 60% of the air-saturation value in controls and test vessels.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

10; 50; 100 and 1000 mg test item.L-1 nominal loading rates.

Details on test conditions:

TEST CONDITIONS
A semi-static test was performed with renewal of test solutions after 24 hours (because of the properties of the test item).
All-glass test tubes of approximately 20 mL capacity sealed with screw caps. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and concentration.
4 replicates with daphnids for the control and per loading rates. Moreover, abiotic replicates (for analytical monitoring) were prepared for each treatment (in glass flasks filled with minimum headspace with test solutions and sealed with screw caps; approximate volume: 100 mL) in order to avoid disturbing the daphnids at t=24h and to prevent test item losses on opening test tubes.
Number of Daphnids: 20 per control and test concentration (loading rate), divided into 4 groups of 5 animals
Loading: 5 daphnids per vessel each completely filled with test solution and without headspace

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h light : 8h dark

TEST MEDIUM / WATER PARAMETERS
Reconstituted water, as prescribed by the OECD Guideline 202.
Stock solutions: a) CaCl2.2H2O 11.76 g.L-1
b) MgSO4.7H2O 4.93 g.L-1
c) NaHCO3 2.59 g.L-1
d) KCl 0.23 g.L-1
An aliquot (25 mL) of each solution (a to d) was added to each litre (final volume) of deionised water (conductivity < 10 μS.cm-1).
The pH of this solution was in the range of 6 to 9 and the total water hardness was approximately 250 mg.L-1 (as CaCO3). This reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value, and then set aside for 2 days without aeration.

EFFECT PARAMETERS MEASURED
Immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Immobile animals were eliminated from the vessels as soon as they were discovered. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.

RANGE-FINDING STUDY
- Test concentrations: loading rates of 10, 50, 100 and 1000 mg.L-1
- Results used to determine the conditions for the definitive study: After 48 hours of exposure, immobilisations were 0% at 10 and 50 mg.L-1 (loading), 30% at 100 mg.L-1 (loading) and 100% for the loading rate 1000 mg test item.L-1.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of test item

Basis for effect:

mobility

Details on results:

After 24 hours of exposure, immobilisations were 0% at 10 and 50 mg.L-1 (loading), 10% at 100 mg.L-1 (loading) and 60% at 1000 mg.L-1 (loading). After 48 hours of exposure, immobilisations were 0% at 10 and 50 mg.L-1 (loading), 25% at 100 mg.L-1 (loading) and 100% at 1000 mg.L-1 (loading). It should be noted that after 24 and 48 hours of exposure some residual particles were lying at the bottom of the test tubes at the nominal loading rates of 50 to 1000 mg.L-1 (more visible at the highest LR). These particles could be the cause of the observed immobilisations on daphnids (physical effect) rather than toxicity due to the test substance, but regarding results of TOC content in WAFs an effect directly due to a test item effect is not excluded. However, this should be taken cautiously because the test item was probably in excess and thus some constituents may have been above their solubility limit.
Based on these results (very close after 48h to those of the range-finding test), the 48-hour EC50 was therefore > 100 mg test item.L-1 (loading).

Results with reference substance (positive control):

On April 10, 2015 (most recent test at this time), the 24h-EC50 was 1.01 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with the OECD 202 (expected 24h-EC50: 0.6 mg.L-1 to 2.1 mg.L-1) at this time.

Reported statistics and error estimates:

No data.

Validity criteria

Controls: In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress.

Dissolved [02]: Dissolved oxygen concentration at the end of the test was ≥ 60% of the air-saturation value in controls and test vessels. Thus the validity criteria have been fulfilled in the present study.

Water quality parameters and environmental conditions throughout the test

pH and oxygen concentrations (mg.L-1) remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen: ≥ 60% of the air-saturation value at the end of the test). It should be noted that a decrease in pH values was observed in particular at the highest WAFs concentrations (in comparison with pH values of the control).

Furthermore, the temperature of the test medium was situated between 20.6 and 21.3 °C throughout the test (average value: 20.9°C), and complied with the requirements as laid down in the study plan (20°C ± 2°C, constant within 1°C).

Analytical results

Concentration of dissolved organic material in the WAFs was checked by analysis of total organic carbon (TOC) in the control medium and the WAFs at the start, at t=24h (new and old solutions) and at the end of the test. Based on the results of Total Carbon (TC ) and Total Inorganic Carbon (IC) measurements the TOC content of the test samples was determined using the following equation: TOC = TC – IC.

Although every effort was made in a first time to extract and solubilize the soluble fraction of the test item in test water (heating, sonication, mixing without headspace …) and secondly to maintain the concentrations of the WAFs (semi-static and closed conditions without headspace), analytical results of this test indicate that unquantifiable or very few organic compounds were found in WAFs,

except at the loading rate of 1000 mg.L-1

It should be noted that chemical analyses were performed only to provide an indication of the concentration of dissolved organic material in the WAFs and their stability. A WAF is by definition a complex mixture for which the individual concentration of each constituent differs due to its properties (e.g. solubility, adsorption, volatilisation, bioaccumulation…). Due to these differences, the ratio of certain constituents may change over time and the composition of the WAFs after 24h will not be exactly that of the fresh solutions. Moreover, interactions between certain constituents of the mixture may occur and affect the behaviour of a given constituent which consequently would not react in the same way that if it was alone in the mixture. Thus, since the study was carried out using WAFs made of several constituents with different stability and behaviours in aqueous solutions during testing, it was considered acceptable to base results on nominal loading values.

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value was found to be higher than 100 mg test item/L.

Executive summary:

A study was performed to assess the acute toxicity of the test item FIR BALSAM RESINOID to Daphnia magna. The method followed was designed to be compliant with the OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23). The criterion measured was the EL50 (Median Effective Loading rate), a statistically derived loading rate which is expected to cause immobility in 50% of test animals within a period of 48 hours.

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rate of 10, 50, 100 and 1000 mg test item.L-1 and to a control. The immobility of the daphnids was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours. Concentration of dissolved organic material in the WAFs was checked by analysis of total organic carbon (TOC) in the control medium and the WAFs at the start, at t=24h (new and old solutions) and at the end of the test. These chemical analyses were performed only to provide an indication of the concentration of dissolved organic material in the WAFs and their stability.

Analytical results showed that unquantifiable or very few organic compounds were found in WAFs, except at the loading rate of 1000 mg.L-1. After 48 hours of exposure, immobilisations were 0% at 10 and 50 mg.L-1 (loading), 25% at 100 mg.L-1 (loading) and 100% at 1000 mg.L-1 (loading). At each observation time, some residual particles were lying at the bottom of the test tubes at the nominal concentrations of 50 to 1000 mg.L-1 (more visible at the highest nominal concentrations). These particles could be the cause of the observed immobilisations on daphnids (physical effect) rather than toxicity due to the test substance.

Under the experimental conditions and based on nominal concentrations, the 48-hour EL50 value estimated was estimated to be higher than 100 mg test item.L-1.

Description of key information

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value was found to be higher than 100 mg test item/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

For that endpoint, a study performed on the registered substance was available. This study aimed to assess the acute toxicity of the test item FIR BALSAM RESINOID to Daphnia magna. The method followed was designed to be compliant with the OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23). The criterion measured was the EL50 (Median Effective Loading rate), a statistically derived loading rate which is expected to cause immobility in 50% of test animals within a period of 48 hours.

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rate of 10, 50, 100 and 1000 mg test item.L-1 and to a control. The immobility of the daphnids was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours. Concentration of dissolved organic material in the WAFs was checked by analysis of total organic carbon (TOC) in the control medium and the WAFs at the start, at t=24h (new and old solutions) and at the end of the test. These chemical analyses were performed only to provide an indication of the concentration of dissolved organic material in the WAFs and their stability.

Analytical results showed that unquantifiable or very few organic compounds were found in WAFs, except at the loading rate of 1000 mg.L-1. After 48 hours of exposure, immobilisations were 0% at 10 and 50 mg.L-1 (loading), 25% at 100 mg.L-1 (loading) and 100% at 1000 mg.L-1 (loading). At each observation time, some residual particles were lying at the bottom of the test tubes at the nominal concentrations of 50 to 1000 mg.L-1 (more visible at the highest nominal concentrations). These particles could be the cause of the observed immobilisations on daphnids (physical effect) rather than toxicity due to the test substance.

Under the experimental conditions and based on nominal concentrations, the 48-hour EL50 value estimated was estimated to be higher than 100 mg test item.L-1

Based on these results, the test item would not be classified as acute 1 to aquatic organisms according to the CLP.

All validity criteria were successful. This study complied with the requirements of the guideline and can therefore be considered as acceptable for that endpoint.