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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 April - 09 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Physical state/appearance: amber coloured liquid
- Batch No.of test material: AN-0400-112
- Expiration date of the lot/batch: 01 January 2019
- Purity: 100% UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition prior to analysis: samples were frozen
- Storage condition of test material: At approximately -20 ºC in the dark, under nitrogen
Analytical monitoring:
yes
Details on sampling:
Not specified
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Method:
- Aqueous media was prepared via mixing the test substance with water for a prolonged period and then the mixture was allowed to settle.
- The test substance phase was separated by siphon.
- Test organisms exposed to the aqueous phase or Water Accomodated Fraction (WAF), which may contain dissolved test substance and/or leachates from the test substance.
- Exposures expressed in terms of the original concentration of the test substance in water at the beginning of the mixing period (loading rate), irrespective of the actual concentration of test substance in the WAF.

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: Derived from in-house laboratory cultures.
- Age of parental stock (mean and range, SD): not specified
- Feeding during test: Yes
- Food type: A mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension
- Amount: not specified
- Frequency: daily

ENVIRONMENTAL CONDITIONS
- Temperature-controlled room
- Water temperature: 18-22°C
- Light/dark cycle: 16 hours light and and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
Test type:
static
Water media type:
other: Reconstitued water
Remarks:
Elendt M7 Medium
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours. Daphnia were considered to be immobilised if they were unable to swim within 15 seconds after gentle agitation.
Hardness:
Elendt M7 medium = 262 mg/K as CaCO3 in the range-finding test and 246 mg/L as CaCO3 in the definitive test
Test temperature:
22°C
pH:
7.9 ± 0.3
Dissolved oxygen:
≥ 3 mg/L
Salinity:
not specified
Conductivity:
not specified
Nominal and measured concentrations:
Range-finding test: nominal amounts of test substance (5.0, 50 and 500 mg) were separately added to the surface of 5 L test water to give loading rates of 1.0, 10 and 100 mg/L, respectively.
Details on test conditions:
TEST SYSTEM
- Test vessel: 150mL glass beakers containing Elendt M7 medium
- Type: closed to reduce evapouration
- Reproduction: culture conditions ensured reproduction was via parthenogenesis.
- Renewal rate of test solution (frequency/flow rate): test solutions were not renewed during the exposure period
- No. of organisms per vessel: 5 daphnids placed in each test and control vessel at random in the test solutions
- No. of vessels per concentration (replicates): four replicate test vessels
- No. of vessels per control (replicates): four control vessels
- Gravid adults are isolated the day prior to the start of the test so young daphnids produced overnight were less than 24 hours old.
- The young daphnids were removed from the cultures and used for testing - no first brood progeny used

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods

VEHICLE CONTROL PERFORMED: no

POSITIVE CONTROL PERFORMED: yes
- maintained under identical conditional but not exposed to the test subtance (untreated Elendt M7 media)

RANGE-FINDING STUDY
- Test concentrations: nominal amounts of test substance (5.0, 50 and 500 mg) were separately added to the surface of 5 L test water to give loading rates of 1.0, 10 and 100 mg/L, respectively.
- Results used to determine the conditions for the definitive study: No immobilisation observed at 1.0, 10 and 100 mg/L loading rate WAF, there fore a single loading rate (limit test) of four replicates, of 100 mg/L (nominal amount of test susbtance = 500 mg), was selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
42 h
Dose descriptor:
NOELR
Effect conc.:
ca. 100 mg/L
Nominal / measured:
nominal
Basis for effect:
mobility
Remarks on result:
other:
Remarks:
After 24 and 48 hours exposure
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Details on results:
RANGE-FINDING TEST:
- No immobilisation was seen at 1.0, 10 and 100 mg/L loading rate WAF.
- Some trapping of the daphnia was observed in all preparations, but this was considered to not have had an signficant effect on the outcome of the test.
>Chemical analysis of test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained. It was determined to be 0.021 mg/L. This does not suggest that no test susbtance was in the solution, but that any dissolved test substance was at a concentration less than the LOQ.

DEFINITIVE TEST:
> Chemical analysis of test preparations at 0 and 48 hours showed test concentrations of less than LOQ, determined to be 0.021 mg/L. As with the range-finding test, this does not conclude that no test substance was present in the solution, but rather any dissolved test substance was present at a concentration less than LOQ.
- The dissolved test substance may have been one or several components of the test substance and since toxicity can only be attributed to the test substance as a whole, the results were based on nominal loading rates only.

> Immobilisation data
- no immobilisation seen in 20 daphnids exposed to 100 mg/L loading rate WAF for 48 hours
- it was considered unnecesary and unrealistic to test at loading rates more than 100 mg/L.

> Sub-lethal effects
- No sub-lethal effects were observed throughout the test.

> Water quality critera
- temperature was maintained at 22°C daily and there were no treatment-related differences for oxygen concentration or pH.
- light intensity was observed to be 487-521 lux throughout the test.

> Vortex depth measurements
- vortex depth recorded at the start and end of the mixing period was obsreved to be a dimple on the water surface on both occasions.

> Test substance solubility
- at the start of the mixing period: clear colourless water column with an oily layer of test substance on the surface.
- after 23 hours stirring and 1 hour standing period: clear colourless water column with an oily layer of test substance on the surface.
- microscopic examination of WAF showed no micro-dispersions or undissolved test substance to be present
- at the start and throughout the test, all control and test solutions were observed to be clear colourless solutions
Results with reference substance (positive control):
The results from the positive control were within the normal range for the reference substance.

> Validity criteria
- the test was considered valid since no more than 10% of the control daphnids showed immobilisation (actural value 5%) or other signs of disease or stress (e.g. discolouration, unusual behvaiour such as trapping at the surface water)
- the oxygen concentration at the end of the test was ≥ 3mg/L in the control and test vessels (actual value 8.7 mg/L).
Reported statistics and error estimates:
Validaion of the analytical method - linearity: The data was found to have a quadratic correlation within the calibration range of 0 to 15.9 mg/L. The R² fit of the calibration curve to the data was 0.9977, and was considered to be acceptable

Analytical investigations

- the test substance gave a chromatographic profile consisting of multiple peaks

- the detection system was found to have acceptable correlation to the fit of the quadratic equation

- the analytical producedure had acceptable recoveries of test substance in test medium and so the method of analysis was validated and proven suitable for use.

Validity criteria fulfilled:
yes
Conclusions:
Based on the results of this study, the acute toxicity of the test substance, Terpenes and terpenoids, turpentine oil, beta-pinene fraction polymerised, to the freshwater invertebrate Daphnia magna has been investigated and gave 48-Hour EL50 values of >100 mg/L loading rate WAF The No-Observed-Effect-Loading-rate (NOELR) was 100 mg/L loading rate WAF.
Executive summary:

The acute toxicity of the test substance, Terpenes and terpenoids, turpentine oil, beta-pinene fraction polymerised, was assessed to Daphnia magna Straus, following a method compatible with OECD Guidelines for Testing of Chemicals (April 2004) no. 202, 'Daphnia sp. Acute Immobilisation Test' referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Due to the low aqueous solubility and complex nature of the test substance, the test medium was prepared as a Water Accomodated Fraction (WAF). During a preliminary range-finding test, Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L corresponding to nominal amounts of 5.0, 50 and 500 mg test substance, which were each added separately to the surface of 5 litres of test water (Elendt M7). The solution was stirred with a magnetic stirrer. for 23 hours followed by a standing period for 1 hour. Microscopic observation made on the WAFs indicated that a signficant amount of dispersed test substance was present in the water column, thus justifying the removal of WAFs via filtering through a glass wool plug (2 -4cm in length). WAF was removed by mid-depth siphoning, after discarding the first 75 -100 mL, in order to give the loading rate WAFs. Two replicates per concentration were prepared, each containg 5 daphnids. The control group were maintained under identical conditions but were not exposed to the test substance, i.e. untreated Elendt M7 media. The positive control used was potassium dichromate.

Based on the results of the range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a WAF of the test substance at a nominal loading rate of 100 mg/L for 48 hours at a temperature of 22°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ), which was determined to be 0.021 mg/L in the range-finding and definitive test. This did not signify that no test substance was present in the solution, but that dissolved test substance was present at concentrations of less than the LOQ. The dissolved test substance may have been present as one of several components of the test substance and seeing that toxicity can only be attributed to the test substance as a whole, the results were based on nominal loading rates only. The validity criteria of the study was met.

Exposure of Daphnia magna to the test substance gave EL50 values of >100 mg/L loading rate WAF and the no-observed-effect-loading rate (NOELR) was 100 mg/L loading rate WAF. It was therefore considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Description of key information

The acute toxicity of the test substance, Terpenes and terpenoids, turpentine oil, beta-pinene fraction polymerised, was assessed to Daphnia magna Straus, following a method compatible with OECD Guidelines for Testing of Chemicals (April 2004) no. 202, 'Daphnia sp. Acute Immobilisation Test' referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Due to the low aqueous solubility and complex nature of the test substance, the test medium was prepared as a Water Accomodated Fraction (WAF). During a preliminary range-finding test, Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L corresponding to nominal amounts of 5.0, 50 and 500 mg test substance, which were each added separately to the surface of 5 litres of test water (Elendt M7). The solution was stirred with a magnetic stirrer. for 23 hours followed by a standing period for 1 hour. Microscopic observation made on the WAFs indicated that a signficant amount of dispersed test substance was present in the water column, thus justifying the removal of WAFs via filtering through a glass wool plug (2 -4cm in length). WAF was removed by mid-depth siphoning, after discarding the first 75 -100 mL, in order to give the loading rate WAFs. Two replicates per concentration were prepared, each containg 5 daphnids. The control group were maintained under identical conditions but were not exposed to the test substance, i.e. untreated Elendt M7 media. The positive control used was potassium dichromate.

Based on the results of the range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a WAF of the test substance at a nominal loading rate of 100 mg/L for 48 hours at a temperature of 22°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ), which was determined to be 0.021 mg/L in the range-finding and definitive test. This did not signify that no test substance was present in the solution, but that dissolved test substance was present at concentrations of less than the LOQ. The dissolved test substance may have been present as one of several components of the test substance and seeing that toxicity can only be attributed to the test substance as a whole, the results were based on nominal loading rates only. The validity criteria of the study was met.

Exposure of Daphnia magna to the test substance gave EL50 values of >100 mg/L loading rate WAF and the no-observed-effect-loading rate (NOELR) was 100 mg/L loading rate WAF. It was therefore considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information