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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:
26 June - 15 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
trans-cinnamic acid
EC Number:
205-398-1
EC Name:
trans-cinnamic acid
Cas Number:
140-10-3
Molecular formula:
C9H8O2
IUPAC Name:
3-phenylacrylic acid
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor / 20171106
- Expiration date of the lot/batch: 10 April 2020
- Purity test date: 09/04/2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in dark over silica gel
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
- Sampling method:
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis.

Preparation of Calibration Standards
The test item (nominal 100 mg) was dissolved in acetonitrile (100 mL) to prepare a stock solution with a nominal concentration of 1000 mg/L. This stock solution was further diluted with acetonitrile:water:formic acid (50:50:0.1 v/v/v) to obtain a nominal 5 mg/L calibration standard. A duplicate calibration standard was similarly prepared at 5 mg/L. These duplicate calibration standards were used to determine the recovery and test sample concentrations.

Preparation of Linearity Standards
The test item (nominal 100 mg) was dissolved in acetonitrile (100 mL) to prepare a stock solution with a nominal concentration of 1000 mg/L. Defined volumes of this stock solution were diluted with acetonitrile:water:formic acid (50:50:0.1 v/v/v) to obtain standards in the range of 0.10 to 10 mg/L. A second standard was similarly prepared at a nominal concentration of 5 mg/L. These standards were used to evaluate the linearity of the analytical system.

Preparation of Test Samples
The test samples were thawed with the aid of a water bath, formic acid added (0.05 mL/50 mL) plus water (50 mL) then diluted with acetonitrile. They were further diluted (if required) with acetonitrile:water:formic acid (50:50:0.1 v/v/v).

Preparation of Accuracy and Precision Samples
To demonstrate the validity of the analytical procedure, volumes of test medium were spiked with the test item and the recovery was assessed. The test item (nominal 100 mg) was initially dissolved in acetonitrile to prepare a stock solution with a concentration of 1000 mg/L. A defined volume of this stock solution was diluted with test medium to obtain spiked recovery samples at a concentration of 1.0 mg/L. Five replicates at this concentration level were prepared and subjected to the same treatment as the test samples. In addition, test medium without the addition of the test item (synthetic control) was also analyzed.

Test solutions

Vehicle:
no
Details on test solutions:
A nominal amount of test item (200 mg) was dissolved in test water and the volume adjusted to 2 liters to give the 100 mg/L test concentration. This test concentration was sonicated for 30 minutes to ensure all of the test item had dissolved after which a series of dilutions was made to give further test concentrations of 1.0, 3.2, 10 and 32 mg/L. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24 and 48 hours

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia Magna
- Strain/clone: Derived from in house culture
- Justification for species other than prescribed by test guideline: N/A

ACCLIMATION
Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7
medium in a temperature controlled room maintaining the water temperature at
18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness
cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a
mixture of algal suspension (Desmodesmus subspicatus) and GM300 feed suspension.
Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were
isolated the day before initiation of the test, such that the young daphnids produced overnight
were less than 24 hours old. These young were removed from the cultures and used for
testing. The diet and diluent water are considered not to contain any contaminant that would
affect the integrity or outcome of the study.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
293.8 mg/l CacL2.2H2O
Test temperature:
20-22°C
pH:
7.9 ±0.3
Dissolved oxygen:
8.2-9.1 mg O2/L
Salinity:
not applicable
Nominal and measured concentrations:
Nominal test concentration: 1.0, 3.2, 10, 32, 100 mg/L
Geometric mean measured test concentrations were determined to be 0.33, 2.3, 8.5, 29 and 94 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass beakers
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 100 mL test preparation
- Volume of solution: 100 mL
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): semi-static; test media renewal at 24 hours; the test concentrations were freshly prepared and the daphnids transferred by wode bore pipettes from the 24-hour old test media into the new media
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
Ingredient Final Concentration (mg/L)
H3BO3 0.715
MnCl2.4H2O 0.090
LiCl 0.077
RbCl 0.018
SrCl2.6H2O 0.038
NaBr 0.004
Na2MoO4.2H2O 0.016
CuCl2.2H2O 0.004
ZnCl2 0.013
CoCl2.6H2O 0.010
KI 0.0033
Na2SeO3 0.0022
NH4VO3 0.00058
Na2EDTA.2H2O 0.625
FeSO4.7H2O 0.249
CaCl2.2H2O 293.8
NaHCO3 64.8
MgSO4.7H2O 123.3
Na2SiO3.9H2O 10
KCl 5.8
NaNO3 0.274
K2HPO4 0.184
KH2PO4 0.143
Thiamine hydrochloride 0.075
Cyanocobalamine (vitamin B12) 0.0010
D(+) biotin (vitamin H) 0.00075

OTHER TEST CONDITIONS
- Adjustment of pH: While there were no treatment related differences for oxygen concentration, concentration dependent differences in pH were observed throughout the test.
- Photoperiod: 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Light intensity: Throughout the test the light intensity was observed to be in the range 907 to 960 Lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24 hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.
nominal amount of test item (50 mg) was dissolved in test water and the volume adjusted to 500 mL to give the test concentration at 100 mg/L. The test concentration was sonicated for 30 minutes to ensure the test item had fully dissolved after which a series of dilutions was made to give further test concentrations of 0.10, 1.0 and 10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test five daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C and a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Two replicate test and control vessels were prepared. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.
The control group was maintained under identical conditions but not exposed to the test item. A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.
- Results used to determine the conditions for the definitive study: Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1.
No immobilization was observed at the test concentration of 0.10 mg/L; however, 10% immobilization was observed at 1.0 mg/L, 30% immobilization was observed at 10 mg/L and 100% immobilization was observed at 100 mg/L.
Reduced mobility was noted in one Daphnia at a test concentration of 100 mg/L following 24 hours exposure.

Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the LOQ, determined to be 0.031 mg/L, to 97 mg/L. There was a concentration dependent decline in the measured concentrations at 48 hours in the range of less than the LOQ to 91 mg/L indicating that the test item was unstable under the conditions of the test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
8.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
29 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Other biological observations: Sub-lethal effects of exposure were observed in measured test concentrations of 2.3, 29 and 94 mg/L. These responses were reduced mobility, covered in debris and trapping at surface

Any other information on results incl. tables

Verification of Test Concentrations

Analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 87% to 95% of their nominal concentrations except at the lowest test level of 1.0 mg/L, where the measured levels were 68% and 67% of nominal, respectively. Analysis of the old or expired test preparations at 24 and 48 hour confirmed that the initial measured test concentrations at 10 to 100 mg/L were maintained, ranging from 78% to 95% of their nominal concentrations. At the two lowest test concentrations of 1.0 and 3.2 mg/L, the measured concentrations had declined, with values ranging from less than the limit of quantification (LOQ; determined to be 0.031 mg/L) to 70% of nominal. Given this decline it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data.

Immobilization Data

Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2 (attached). The relationship between percentage immobilization and concentration at 48 hours is given in tfigure 1 of the test report.

Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression based on the geometric mean measured test concentrations gave the following results:

Time (hr)

EC50

(mg/L)

95% confidence limits

24

>94

Not determined due to the nature of the data

48

32

The NOEC after 24 hours exposure was considered to be 29 mg/L given that 30% immobilization was observed in the 94 mg/L test concentration. Correspondingly the LOEC after 24 hours exposure was considered to be 94 mg/L. The NOEC after 48 hours exposure was 8.5 mg/L. The LOEC after 48 hours exposure was 29 mg/L. The slopes and their standard errors of the response curves at 24 and 48 hours were 8.1 (standard error = 0.35) and 1.1 (standard error = 0.15) respectively.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item gave the following results based on the geometric mean measured test concentrations (geometric mean used as a worst-case):
EC50 (48h): 32 mg/L
NOEC (48h): 8.5 mg/L
LOEC (48h): 29 mg/L
Executive summary:

SUMMARY

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the 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.

Methods

Following a preliminary range-finding test and initial experiment, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L for 48 hours at a temperature of 20°C to 22°C under semi-static test conditions. The number of immobilized Daphnia were recorded after 24 and 48 hours.

Results

Analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 87 to 95% of their nominal concentrations except at the lowest test concentration of 1.0 mg/L where the measured levels were 68% and 67% of nominal, respectively. Analysis of the old or expired test preparations at 24 and 48 hour confirmed that the initial measured test concentrations at 10 to 100 mg/L had been maintained, ranging from 78% to 95% of their nominal concentrations. At the two lowest test concentrations of 1.0 and 3.2 mg/L, the measured concentrations had declined, with values ranging from less than the limit of quantification (LOQ; determined to be 0.031 mg/L) to 70% of nominal. Given this decline it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The geometric mean measured test concentrations were determined to be 0.33, 2.3, 8.5, 29 and

94 mg/L. Exposure of Daphnia magna to the test item gave the following results based on the geometric mean measured test concentrations:

EC50 (48h): 32 mg/L

NOEC (48h): 8.5 mg/L

LOEC (48h): 29 mg/L