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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:
2017-04-04 to 2017-06-02
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Name (as stated in the report): Silvial
Batch No.: SC00017272
Expiration date: 2017-10-13
Chemical Name: 2-METHYL-3-(4-(2-METHYLPROPYL)PHENYL)PROPANAL
Purity: 99.0%
Analytical monitoring:
yes
Details on sampling:
The concentrations were determined via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and at the renewal of the test solutions (24 hours) and in old media at the renewal (24 hours) and at the end of the exposure (48 hours) of all tested dilutions of the saturated solution and the control.
The samples of all tested concentrations were stabilized factor 2 with acetonitrile containing 0.2% H3PO4 after sampling and analyzed without further dilution. All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.
Vehicle:
no
Details on test solutions:
A number of non-GLP preliminary rangefinder tests were conducted using different methods of preparation of the test item in efforts to obtain as much of the parent substance in the test medium at the start of the test. The saturated solution approach and water-miscible solvent method were both explored. For comparison please see acute algae study with test item (Klix, 2017). In summary, the use of solvent resulted in dispersions containing droplets of undissolved test item and gave measured concentrations that were higher in the 50 mg/L nominal test solution than in the 100 mg/L nominal test solution, which was possibly a result of the fact that the substance didn’t stay in solution after application of the stock solution in acetone in algal medium. In contrast, the saturated solution approach gave a series of test solutions with the expected increasing trend in measured concentrations and was therefore considered to be the most appropriate method of preparation of the test item.

A saturated solution with a nominal concentration of 100 mg/L of the test item, which corresponds to 106.8 pL/L of the test item (specific gravity of 0.9362 (20/20 °C) taken into account), was prepared one day prior to the start of the exposure (at -24 hours) and the water renewal (0 hours) in a glass flask.
The glass flask was filled up with 1150 mL of the dilution water, 122.8 pL of the test item was placed onto the water surface and the bottle was closed with a screw cap. A small headspace of a few milliliters was accepted. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at room temperature for 24 hours. After completion of stirring, the saturated solution was allowed to stand for approximately 1 hour for separation of undissolved test item. The saturated solution was removed by siphoning from the approximate bottom of the glass flask. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of emulsion). No undissolved test item was detected. The saturated solution was used as a stock solution for the preparation of the definitive test concentrations by dilution with dilution water.
Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnia magna STRAUS (Clone 5).
Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
Culture: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx
Culture medium: Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaC03/L, is used.
Feeding of the culture stocks: The daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 10s cells/mL. The algae were cultured at the test facility.
Age of the daphnids at the start of the exposure: Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
166 - 174 mg CaC03/L
Test temperature:
18-22 °C, constant within ± 1 °C
pH:
7.47 - 7.75
Dissolved oxygen:
8.84 - 9.12 mg/L
Conductivity:
430 - 467 microS/cm
Nominal and measured concentrations:
The undiluted saturated solution and four dilution levels prepared out of the saturated solution in a geometric series with a separation factor of 2.2 by dilution of the saturated solution with dilution water, were tested as follows: 4.27 - 9.39 - 20.7 - 45.5 - 100% of the saturated solution. Those values correspond to geometric mean measured concentrations of 0.600 - 0.736 - 1.80 - 4.26 - 11.8 mg/L (Sum of the Analytes : Parent and Metabolite). Values < LOQ were taken with half of the LOQ = 0.600 mg/L into account for the calculation of the geometric mean measured concentrations
Details on test conditions:
Due to the volatile nature of the test item, a closed system (closed test vessels without headspace) was chosen for testing according to OECD guidance document no. 23, ENV/JM/MONO(2000)6 to reduce contact with air and losses of the test item by evaporation. Based on the results of the non-GLP preliminary range finding tests, the study was performed with a semi-static test design under diffuse light conditions.
Test vessels / volume: Sealed glass flasks (4.5 (ID) x 9.5 (H) cm) with screw cap were used and filled up to the top with the test solutions. A test volume of approximately 130 mL was provided in each test vessel.
Number of daphnids and replicates: 20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for each concentration level and the control. The daphnids were not fed during the study.
The test vessels were filled up to the top with the test solutions. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette. Thereafter, the test vessels were closed immediately with screw caps
Reference substance (positive control):
yes
Remarks:
Potassium dichromate p.a. (Sigma) purity 99%
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
4.71 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility

The study was performed according to OECD Guideline 202 (2004). The validity criteria were fulfilled:

• In the control group, no daphnids were immobilized or showed any signs of disease or stress, e.g. discoloration or unusual behavior such as trapping on the surface of the water, during the 48-hour test period (required: not more than 10% of the daphnids immobilized in the control).

• The dissolved O2 concentration in the 24-hours old media was > 7.02 mg/L (required: > 3 mg/L in the 24-hours old media) in all test vessels of all tested concentration levels and the control.

Validity criteria fulfilled:
yes
Remarks:
The validity criteria of the test guidelines were fulfilled.
Conclusions:
Based on the geometric mean measured concentrations of the test item Silvial, the 48 hour-EC50 for Daphnia magna was 4.71 mg/L (95% confidence limits: 4.27 - 9.93 mg/L).
Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Silvial (batch number: SC00017272) were determined at the test facility according to OECD 202 (2004) from 2017-04-04 to 2017-06-02, with the definitive exposure phase from 2017-05-31 to 2017-06-02.

The test item was found to be rapidly degraded. Therefore, a number of non-GLP preliminary rangefinder tests were conducted using different methods of preparation of the test item in efforts to obtain as much of the parent substance in the test medium at the start of the test. The saturated solution approach and water-miscible solvent method were both explored. For comparison please see acute algae study with test item (Klix, 2017). In summary, the use of solvent resulted in dispersions containing droplets of undissolved test item and gave measured concentrations that were higher in the 50 mg/L nominal test solution than in the 100 mg/L nominal test solution, which was possibly a result of the fact that the substance didn’t stay in solution after application of the stock solution in acetone in algal medium. In contrast, the saturated solution approach gave a series of test solutions with the expected increasing trend in measured concentrations and was therefore considered to be the most appropriate method of preparation of the test item. One degradation product of the test item was determined in all samples. The degradation product was confirmed to be 3-(4-isobutylphenyl)-2- methylpropanoic acid by comparison of the peaks observed in the chromatograms from the preliminary range-finder tests with a reference standard provided by the sponsor.

The study was conducted in a closed system without headspace under semi-static conditions over a period of 48 hours with 5 concentrations of the test item Silvial in the range of 4.27 to 100% of the saturated solution, prepared in a geometric series with a separation factor of 2.2.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control.

The concentrations of Silvial were determined via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and at the renewal of the test solutions (24 hours) and in old media at the renewal (24 hours) and at the end of the exposure (48 hours) of all tested dilutions of the saturated solution and the control. The concentrations of the degradation product were analytically verified in addition to the concentrations of the parent substance.

The degradation product of Silvial was determined in all samples and converted into test item concentrations with the ratio of molar weights. The measured concentrations of the test item (sum of the test item and the calculated concentration converted to the degradation product) at the end of the intervals were < LOQ to 118% of the initially measured values at the start of the intervals (0 and 24 hours).

Using the sum of the measured concentrations of the parent substance and the calculated concentration based on the measured concentration converted to the degradation product, the geometric mean measured concentration of the test item was calculated. These were determined to be 0.600 - 0.736 - 1.80 - 4.26 - 11.8 mg/L. Values < LOQ were taken with half of the LOQ = 0.600 mg/L into account for the calculation of the geometric mean measured concentrations. For results, see Table 9 to Table 11.

Based on the geometric mean measured concentrations of the test item Silvia, the 48 hour-EC50 for Daphnia magna was 4.71 mg/L (95% confidence limits: 4.27 - 9.93 mg/L).

Description of key information

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Silvial (batch number: SC00017272) were determined at the test facility according to OECD 202 (2004) from 2017-04-04 to 2017-06-02, with the definitive exposure phase from 2017-05-31 to 2017-06-02.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
4.71 mg/L

Additional information

The study was conducted in a closed system without headspace under semi-static conditions over a period of 48 hours with 5 concentrations of the test item Silvial in the range of 4.27 to 100% of the saturated solution, prepared in a geometric series with a separation factor of 2.2. The saturated solution method of preparation for the test item was considered to be the most appropriate following media preparation trials performed as part of an algal inhibition study (KLIX, 2017). Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control.

The concentrations of Silvial were determined via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and at the renewal of the test solutions (24 hours) and in old media at the renewal (24 hours) and at the end of the exposure (48 hours) of all tested dilutions of the saturated solution and the control. The concentrations of the degradation product were analytically verified in addition to the concentrations of the parent substance.

The degradation product of Silvial was determined in all samples and converted into test item concentrations with the ratio of molar weights. The measured concentrations of the test item (sum of the test item and the calculated concentration converted to the degradation product) at the end of the intervals were < LOQ to 118% of the initially measured values at the start of the intervals (0 and 24 hours).

Since the biological effects observed are produced by the mixture of parent substance and metabolite, it is considered justifiable to base all effect values on the geometric mean measured test item concentrations of Silvial (parent substance and the calculated concentration of parent based on the measured concentration converted to the degradation product), which were calculated to be: 0.600 - 0.736 - 1.80 - 4.26 - 11.8 mg/L. Values < LOQ were taken with half of the LOQ = 0.600 mg/L into account for the calculation of the geometric mean measured concentrations.

Based on the geometric mean measured concentrations of the test item Silvial, the 48 hour-EC50 for Daphnia magna was 4.71 mg/L (95% confidence limits: 4.27 - 9.93 mg/L).