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Ecotoxicological information

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:
From 2017-12-22 to 2018-04-12
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 guidelineopen allclose all
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: The concentration of TRIISONONYLAMINE was analyzed in one of the duplicate test medium samples from the single loading rate of 100 mg/L and the control at the start and end of each test medium renewal period.
- Sampling method: For the determination of the actual test item concentration, duplicate samples were taken from the single test concentration and the control at the start and at the end of the two 24-hour test medium renewal periods. For sampling from the aged test medium, the contents of the respective replicates were combined prior to sampling.
- Sample storage conditions before analysis: Immediately after sampling, methanol + 0.2% formic acid (10 mL methanol + 0.2% formic acid per 10 mL sample volume) was added to each sample to stabilize the latter during the storage period. Thereafter, all samples were deep-frozen (at about -20 °C). Based on pre-experiments for investigation of the storage stability, the test item was found to be stable in the test water under these storage conditions.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Pre-Experiment for Test Medium Preparation:
To determine the solubility of the poorly soluble test item (water solubility < 100 mg/L) and the optimum time period to reach equilibration between dissolved and undissolved test item within a reasonable time period, the following stirring pre-experiment was performed:
As the test item is a liquid with low water solubility (<100 mg/L), the slow-stirring method was applied for preparation of an equilibrated test item solution. The slow-stirring method is an alternative method for preparing equilibrated solutions for insoluble, liquid test items, which are suspected to pass filters
during filtration process. In this stirring experiment the test item was carefully applied onto the surface of the test water in the almost completely filled stirring vessel at a loading rate of 100 mg/L without mixing the test item into the water column. Then, the stirring vessel was tightly sealed with glass stoppers to avoid evaporation of the volatile test item. A small headspace in the mixing vessel had to be included for practical reasons as the test item was floating on the water surface. Then, slow stirring was started and samples were taken after 48, 72, 96, 120 and 144 hours. The samples were carefully harvested from the lower part of the water column through a tap at the bottom of the of the stirring vessel and as a precaution, the medium was checked by the Tyndall effect for the presence of undissolved test item particles. The upper part in the mixing vessel of the test medium with undissolved test item floating on the surface remained in the stirring vessel and was not used.
These results showed that the maximum concentration of dissolved test item in test water was reached after a stirring period of 96 hours (no significant increase after 96h stirring time). Since the measured concentrations in the samples without preconditioning are identical or even higher to the samples with preconditioning, no additional preconditioning step was applied for the main test. Additionally, an aliquot of the test medium after 48 hours stirring period (measured concentration of 0.092 μg/L) was incubated for 48 hours at room temperature in an open test vessel and in a test vessel completely filled with test medium and closed with a glass stopper (closed system). The test item concentration of the aged test medium in the open vessel was period.
Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test medium was clear solution with no evidence of undissolved test item. The test medium was prepared before the start of the test (i.e., introduction of the daphnids to the test medium) and prior to the test medium renewal after 24 hours.
The preparation of the test medium was based on the OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna Straus
- Source/Age of parental stock: clone of this species (originally from the Daphnia Collection of the University of Basel/Switzerland in 2015) is successfully bred in IES Ltd Laboratories.
- Feeding during test: no
Breeding conditions:
- Food type/Amount/Frequency: fed three times a week with an algal suspension of the green algae Desmodesmus subspicatus, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen/Germany) and cultivated at IES Ltd Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-GmbH, 49324 Melle/Germany).
- Culture medium and conditions: reconstituted water of the quality identical to the water quality used in the tests (with respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests:
Reconstituted test water (ISO Test water) according to OECD Guideline No. 202. The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity. The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, the test water was not aerated.

ACCLIMATION: not needed since water quality is the same between breeding and testing

At the start of the test, the organisms used in the test were 6-24 hours old and were not first brood progeny.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h

Test conditions

Hardness:
250 mg/L as CaCO3
Test temperature:
The test was performed in a temperature controlled room with continuous monitoring of the room temperature. The test water temperature was maintained between 20 and 21 °C.
pH:
7.7-7.9
Dissolved oxygen:
8.0-8.4
Nominal and measured concentrations:
Nominal: single loading rate of 100 mg/L
Mean Measured Concentration of the Test Item during the 48-Hour Test Period: 0.15 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: in glass vessels completely filled (without headspace) with 60 mL test medium and tightly sealed with glass stoppers to avoid losses of the volatile substance by evaporation (closed system).
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): at 24H
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: The volume of test medium provided for each daphnid was 12 mL (60 mL per replicate).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (ISO Test water) according to OECD Guideline No. 202 was used in the study. It consisted of analytical grade salts dissolved in purified water at the following nominal concentrations:
CaCl2 × 2H2O : 2.0 mmol/L; 294 mg/L
MgSO4 × 7H2O 0.5 mmol/L;123 mg/L
NaHCO3 0.75 mmol/L; 65 mg/L
KCl 0.075 mmol/L; 5.8 mg/L
Water Hardness 2.5 mmol/L; 250 as CaCO3 mg/L
Alkalinity 0.8 mmol/L
- Ca/mg ratio: The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity.
The pH of the test water was 7.5.
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH, dissolved oxygen were measured at T0 and 48H. Temperature-controlled room with continuous monitoring of the room temperature.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light to 8-hour dark cycle with a 30-minute transition period.
- Light intensity: between 15 and 17 μmol m-2 s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The immobility of the daphnids was determined by visual inspection after 24 and 48 hours of exposure. Those daphnids not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobilized. Observations were also performed for other non-lethal effects (e.g. discoloration, surface trapping, reduced swimming etc.).

VEHICLE CONTROL PERFORMED: not appropriate

RANGE-FINDING STUDY: Considering the low test item concentrations measured in the stirring experiment the definitive test was performed as limit test without prior rangefinding test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate is tested as a positive control twice a year.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: based on initial loading rate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.15 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
>= 0.15 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Initial loading rate
Key result
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 0.15 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: Observations were also performed for other non-lethal effects (e.g. discoloration, surface
trapping, reduced swimming etc.), but none was observed.
- Mortality and other adverse effects of control: in the control no daphnids showed immobilization or other signs of disease or stress (e.g., discoloration, surface trapping, reduced swimming etc.).
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no, No remarkable observations were made concerning the appearance of the test media. All test media remained clear solutions throughout the test.
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
The result of the latest positive control test in October 2017 (24-hour EC50: 1.3 mg/L) showed that the sensitivity of the test organisms was within the range given by the guideline (24-hour EC50: 0.60-2.1 mg/L).
Reported statistics and error estimates:
The NOEC, EC0, EC50 and EC100 were directly determined from the raw data.

Any other information on results incl. tables

Due to the decrease of test item concentration during the test medium renewal periods of 24 hours, the mean concentration of the test item was calculated as the geometric mean of the test item concentrations measured at the start and the end of each test medium renewal period (i.e. Day 0 to Day 1, Day 1 to Day 2). From the two geometric mean values obtained, the mean measured test item concentration during the test period of 48 hours was calculated as an arithmetic mean.

 

Loading Rate

Analytically Measured Test Item Concentrations

Mean Measured Concentration of the Test Item during the
48-Hour Test Period

Start of the Renewal Periods (Day 0 / Day 1)

End of the Renewal Periods (Day 1/ Day 2)

[µg/L]

[µg/L]

[µg/L]

100 mg/L*

0.153 / 0.199

0.132 / 0.135

0.15

  *:       Equilibrated test medium with a loading rate of 100 mg/L.

Effect of TRIISONONYLAMINE on the Mobility of Daphnia magna

Mean Measured Test Item
Concentration

No. of
Daphnids
Tested

Immobilized
Daphnids after
24 Hours

Immobilized
Daphnids after
48 Hours

[µg/L]

 

No.

[%]

No.

[%]

Control

20

0

0

0

0

0.15

20

0

0

0

0

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
In the control no daphnids showed immobilization or other signs of disease or stress. Furthermore, the dissolved oxygen concentration at the end of the test was ≥3 mg/L in the test vessels of the control and the test item treatment.
Conclusions:
The test item had no acute toxic effects on Daphnia magna in a semi-static 48-hour test at the measured concentration of 0.15 μg/L, corresponding to the solubility limit of the test item in test water at a loading rate of 100 mg/L.
Executive summary:

The acute toxicity of the test item TRIISONONYLAMINE on Daphnia magna was determined in a 48-hour semi-static test according to the OECD Guideline for Testing of Chemicals, No. 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2. and following GLP. A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test organisms up to and including a loading rate of 100 mg/L.

As the test item is a liquid with low water solubility, in order to prepare a saturated test item solution, the slow-stirring method (to avoid formation of micro emulsions) was applied. For preparation of the test medium, the test item with specific gravity of 0.820 was carefully applied (pipetted) onto the surface of the test water at a loading rate of 100 mg/L. Thereafter slowstirring was applied for 96 hours in a closed vessel to reach a maximum concentration of dissolved test item in the test water. The stirring vessel was nearly completely filled (a small headspace had to be included as the test item was floating on the water surface) and was tightly sealed with glass stoppers to avoid losses of the volatile test item by evaporation during stirring.

After this treatment the lower part of the equilibrated test medium was carefully harvested from the stirring vessel through a tap at the bottom of the vessel. This equilibrated aqueous phase with a loading rate of 100 mg/L, containing dissolved test item only was used as test medium. Additionally, a control (test water without test item) was tested in parallel.

At the end of the test medium renewal periods, 87 and 68 % of the initially measured values were found. Mean measured concentrations during the test period was calculated to be 0.15 µg/L.

In the control and at the mean measured concentration of 0.15 μg/L, no immobilized test organisms were observed during the test period of 48 hours. Therefore, the 48-hour NOEC and EC0 of TRIISONONYLAMINE to Daphnia magna were determined to be at least at the mean measured concentration of 0.15 μg/L.

In conclusion, the test item TRIISONONYLAMINE had no acute toxic effects on Daphnia magna in a 48-hour semi-static test up to its solubility limit in test water at a loading rate of 100 mg/L.