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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 2013-June-11 to 2013-August-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal concentrations selected for this study were 1.0, 3.2, 10, 32 and 100 mg/L
Concentration of samples collected in the pretest period were approximately 33 to 92% of nominal concentration.
Concentration of samples collected during the test were approximately 39 to 88% of nominal concentration.

- Sampling method range-finding test: a sample of each test concentration was taken for chemical analysis at 0 and 48 hoursin order to determine the stability of the test item under test conditions. All samples were stored at approximately -20 °C prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

- Sampling method definitive test (verification of test concentrations): Water samples were taken from the control and each test group (replicates R1 – R4 pooled) at 0 and 48 hours for quantitative analysis. Duplicate samples were taken and stored at -20 °C for further analysis if necessary.

- Sample storage conditions before analysis: samples were stored at -20 °C prior to analysis. All test samples were analyzed on the day of receipt.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION FOR RANGE-FINDING TEST

An amount of test item (1100 mg) was added to 11 liters of reconstituted water and stirred using a propeller stirrer at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and as a precautionary measure, any undissolved test item was removed by filtration through a 0.2 µmm Sartorius Sartopore filter (first approximate 1 liter discarded in order to pre-condition the filter) to give a stock solution with a nominal concentration of 100 mg/L. Serial dilutions were performed in reconstitued water to give the remaining nominal test concentrations of 0.1, 1 and 10 mg/L.

Each stock solutions 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 and 48 hours. See appendix 5 for details and results.

PREPARATION AND APPLICATION OF TEST SOLUTION FOR DEFINITIVE TEST
An amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirrer at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and as a precautionary measure, any undissolved test item was removed by filtration through a 0.2 µmm Felman Acropcap filter (first approximate 100 mL discarded in order to pre-condition the filter) to give a stock solution with a 0-Hour measured concentration of 92 mg/L (corrected for a test item purity value of 93.2%). Aliquots (10, 32, 100 and 320 mg) were each separetely added to a final volume of 1 liter to give tge remaining nominal concentrations of 1.0, 3.2, 10 and 32 mg/L respectively.

Each stock solutions 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 and 48 hours. See appendix 5 for details and results

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out with 1st instar Daphnia Magna derived from in-house laboratory cultures.
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.

Test Species
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °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 Tetramin flake food 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.

Test Water:
The reconstituted water used for the definitive test was the same as that used to maintain the stock animals (see Section 'Details on Test Conditions' for details).

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Not applicable

Test conditions

Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.
Test temperature:
Between 20-22 °C.
See Table 3 Physico-chemical measurements for detailed values obtained throughout the test.
pH:
measured at 0 and 48 hours: between 7.7-8.4.
See Table 3 Physico-chemical measurements for detailed values obtained throughout the test.
Dissolved oxygen:
measured at 0 and 48 hours: between 8.9 and 9.7 mgO2/L.
See Table 3 Physico-chemical measurements for detailed values obtained throughout the test.
Salinity:
conductivity < 5 µS/cm
Nominal and measured concentrations:
The measured concentrations followed a concentration dependant pattern with the lower percentage of nominal concentrations being obtained at the lower test concentrations. This effect was considered to be due to the volatile nature of the test item where losses during preparation at the lower concentrations had a greater effect on the measured test concentration. As a result of these variable measured concentrations, it was considered appropriate to base the results on the mean measured test concentrations which were determined to be:

Concentrations as mg a.i./L:
Nominal Concentration: 1.0; Measured Concentration: 0.3 (0h), 0.4 (48h). Mean measured concentration: 0.4 mg/L
Nominal Concentration: 3.2; Measured Concentration: 1.7 (0h), 1.6 (48h). Mean measured concentration: 1.7 mg/L
Nominal Concentration: 10; Measured Concentration: 7.0 (0h), 6.7 (48h). Mean measured concentration: 6.8 mg/L
Nominal Concentration: 32; Measured Concentration: 26.2 (0h), 25.3 (48h). Mean measured concentration: 26 mg/L
Nominal Concentration: 100; Measured Concentration: 92.1 (0h), 88.2 (48h). Mean measured concentration: 90 mg/L
Details on test conditions:
TEST SYSTEM
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4

TEST SYSTEM:
In the definitive test completely filled and sealed conical flasks containing approximately 160 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test anc control vessel at random, in the test preparation.

Four replicate test and control vessels were prepared. The test vessels were then maintained in a temperature controlled room at 20°C to 22 °C with a photoperiod of 16 hours light (605 to 613 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item.

The test preparations were not renewed during the exposure period. Any immobilization or adverse reactions were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation


Test water. Reconstituted water used for both range finding and definitive tests were:
i) Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l

ii) Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: 6.8-26 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
6.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Details on results:
Range finding test:
Cumulative immobilization data from the exposure of Daphnia Magna to the test item during the range-finding test are given in table 1 attached.
No immobilization was observed at the test concentrations of 0.1 and 1.0 mg/L. However, immobilization was observed at 10 and 100 mg/L.
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 prepartion (see appendix 5) at 0 hours showed tgat measured test concentration of between 64 and 97% of nominal were obtained and 48 hours showed that measured concentrations of between 45 and 77% were obtained.

Definitive test:
Analysis of the test preparations (see appendix 5) at 0 hours showed that measured concentrations of between 33% and 92% of nominal were obtained and at 48 hours showed that measured test concentrations of between 39% and 88% of nominal were obtained. The measured concentrations followed a concentration dependant pattern with the lower percentage of nominal concentrations being obtained at the lower test concentrations. This effect was considered to be due to the volatile nature of the test item where losses during preparation at the lower concentrations had a greater effect on the measured test concentration. As a result of these variable measured concentrations, it was considered appropriate to base the results on the mean measured test concentrations.

Immobilization data
Cumulative immobilization data from the exposure of Daphnia Magna to the test item during the definitive test are detailed below. The relationship between percentage immobilization and concentration at 24 and 48hours is given in figures 1&2 attached.

Analysis of the immobilization data by the geometric mean method at 24 and 48 hours based pm the mean measured concentrations gave the following results:

Time point 24h:
EC50 13 mg/L
95% confidence limits: 6.8 - 26 mg/L

Time point 48h:
EC50 13 mg/L
95% confidence limits: 6.8 - 26 mg/L

The No Observed Effect Concentration after 24 hours and 48 hours exposure was 6.8 mg/L. The Lowest Observed Effect Concentration after 24 and 48 hours exposure was 26 mg/L.

Validation criteria:
The test was considered to be valid given that none more of the control daphnids showed immobilization or other signs of disease or stress that and that the oxygen concentration at the end of the test was => 3 mg/L in the control and test vessels.

Observations on test item solubility:
Throughout the test the test preparations were observed to be clear colorless solutions.

Physico-chemical Measurements:
The results of the physico-chemical measurements are given in table 3 attached. Temperature was maintained at approximately 21 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Results with reference substance (positive control):
- Results with reference substance valid? Yes

- EC50/LC50:
A positive control (Harlan Study Number 41301833) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8, 3.2 mg/L.

Exposure conditions for the positive control were similar to those in the definitive test.

Analysis of the immobilization data by the trimmed Sparman-Karber method (Hamilton et al 1977) at 24 and 48 hours based on the nominal test concentrations gave the following results:
time point 24 hours: EC50 (mg/L) 1.0; 95% Confidence limits 0.91 - 1.2 mg/L; NOEC 0.56 mg/L; LOEC 1.0 mg/L
time point 48 hours: EC50 (mg/L) 0.71; 95% Confidence limits 0.65 - 0.76 mg/L; NOEC 0.32 mg/L; LOEC 0.56 mg/L

The No Observed Effect Concentration (NOEC) is based upon zero immobilisation at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.

Any other information on results incl. tables

Cumulative Immobilization data in the range -finding test: see tables 1&2 attached

Cumulative Immobilization data in the definitive test: see tables 1&2 attached

Physico-Chemical Measurements (definitive test): see table 3 attached

Analysis of the immobilization data in the positive control (potassium dichromate): see appendix 2 attached

Pre-study Media preparation trial: see appendix 4 attached

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item has been investigated and gave the following results based on mean measured concentrations:
Time pont 48h
EC50 13 mg/L
95% confidence limits: 6.8 - 26 mg/L
No Observed Effect Concentration (NOEC) after 48 hours exposure was 6.8 mg/L.
The Lowest Observed Effect Concentration after 48 hours exposure was 26 mg/L.
Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test item toDaphnia magna. The method followed was designed to be comptaible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphniasp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods.

Information provided by the sponsor indicated the test item was insoluble in water.

Pre-study solubility work conducted indicated that it was not possible to obtain a testable solutions of the tet item using traditional methods of preparation, e.g. ultrasonication. A pre-study media preparation trial indicated that a prolonged stir method was most appropriate for this test item.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nomminal concentrations of 1.0, 3.2, 10, 32 adn 100 mg/L for 48 hours at a temperature of approximately 21 °C under static conditions. The test item solution was prepared by stirring 100 mg/L of test item in culture emdium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period, as a precautionary measure, any undissolved test item was removed by filtration (0,2 µmm Gelman Acrocap filter, first appromiate 100 mL discarded in order to pre-conditioner the filter) to produce a stock solution woth a mean measured test concentration of 92 mg/L (corrected for a test item puritt value of 93.2%) from which a series of dilutions was made to give the remainder of the test concentrations. The number of immobilized Daphnia were recorded after 24 and 48 hours.

Results.

AAnalysis of the test preparations (see appendix 5) at 0 hours showed that measured concentrations of between 33% and 92% of nominal were obtained and at 48 hours showed that measured test concentrations of between 39% and 88% of nominal were obtained. The measured concentrations followed a concentration dependant pattern with the lower percentage of nominal concentrations being obtained at the lower test concentrations. This effect was considered to be due to the volatile nature of the test item where losses during preparation at the lower concentrations had a greater effect on the measured test concentration. As a result of these variable measured concentrations, it was considered appropriate to base the results on the mean measured test concentrations.