Registration Dossier

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:
2004-07-05 to 2005-02-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 2004
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark until 22 June 2004, thereafter approximately 4°C in the dark.
- Stability under test conditions: The test material was suspected to be unstable in the test media.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: For the purpose of the definitive test the test material was dissolved directly in reconstituted water.

Sampling and analysis

Analytical monitoring:
yes
Remarks:
at 0 and 48h
Details on sampling:
- Sampling method: The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0, 24, and 48 h. Water samples were taken from the control (replicates R1 – R2 pooled) and all the test groups (replicates R1 – R2 pooled) at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media)
and from the stock solutions at 0 and 24 hours (fresh media) for quantitative analysis.
- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary. All samples were acidified immediately after being taken in an attempt to stabilise the test material in the test media prior to analysis.

Other: Water temperature, dissolved oxygen concentrations and pH were recorded daily throughout the test.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: In the range-finding tests an amount of test material (100 mg) was dissolved in reconstituted water, with the aid of ultrasonication for approximately 5 minutes, and the volume adjusted 1 litre to give the 100 mg/L test concentration from which serial dilutions were performed in reconstituted water. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
For the purpose of the definitive test the test material was dissolved directly in reconstituted water. An amount of test material (100 mg) was dissolved in reconstituted water with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 1 litre to give a 100 mg/L stock solution. An aliquot (100 mL) of this stock solution was diluted in a final volume of 1 litre of reconstituted water to give a further stock solution of 10 mg/L. An aliquot (500 mL) of the 10 mg/L stock solution was dispersed in a final volume of 5 litres of reconstituted water to give the 1.0 mg/L test concentration from which aliquots (10, 18, 32, 56, 100, 180, 320 and 560 mL) were each separately dispersed in a final volume of 1 litre of reconstituted water to give the remainder of the test series of 0.010, 0.018, 0.032, 0.056, 0.10, 0.18, 0.32 and 0.56 mg/L respectively.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The test material was suspected to be unstable in the test media. Therefore, in an attempt to minimise losses of test material the test media were freshly prepared at 0 and 24 hours prior to dosing using a semi-static test regime.
- Controls: The control group was maintained under identical conditions but not exposed to the test material.
- Test Water: The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.
-- Reconstituted Water:
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.
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3 .

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain/clone: Daphnia magna-
- Source: 1st instar, derived from in-house aboratory cultures.
- Feeding during test: none


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 21°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 suspension of algae (Chlorella sp.). 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. The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
-
Post exposure observation period:
-

Test conditions

Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3 .
Test temperature:
Temperature was maintained at approximately 20°C, the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
pH:
The reconstituted water had a pH of 7.8 ± 0.2. The pH was measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. There were no treatment related differences for pH.
Dissolved oxygen:
The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value. The test vessels were not aerated. The dissolved oxygen concentration was measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. There were no treatment related differences for oxygen concentration.
Nominal and measured concentrations:
please refer to Any other information on results incl. tables
Details on test conditions:
TEST SYSTEM
- Test vessel: jars
- Type (delete if not applicable): covered to reduce evaporation
- Material, size, headspace, fill volume: 250 mL glass jars containing approximately 200 mL of test preparation
- Aeration: the test vessels were not aerated
- Type of flow-through (e.g. peristaltic or proportional diluter): none
- Renewal rate of test solution (frequency/flow rate): Semi-static test conditions were employed in the test in order to maintain as near nominal test concentrations as possible. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water:
i) Stock Solutions
a) CaCl 2 .2H 2 O 11.76 g/L
b) MgSO 4 .7H 2 O 4.93 g/L
c) NaHCO 3 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 an approximate theoretical total hardness of 250 mg/L as CaCO3 .
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilisation 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 immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

RANGE-FINDING STUDY
- Test concentrations: see below
- Results used to determine the conditions for the definitive study: see below
Reference substance (positive control):
yes
Remarks:
potassium dichromate (Sigma Lot No 21K1444)

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other:
Remarks:
A 24-Hour EC 50 value was not calculated as less than 50% immobilisation occurred at this time point.
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other:
Remarks:
A 24-Hour EC 50 value was not calculated as less than 50% immobilisation occurred at this time point.
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.23 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks on result:
other:
Remarks:
95% Confidence limits: 0.19 - 0.29 mg/l
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.12 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other:
Remarks:
95% Confidence limits: 0.09 - 0.16 mg/l
Duration:
24 h
Dose descriptor:
EC0
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC0
Effect conc.:
0.35 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
0.056 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
0.02 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Mortality of control: no mortality observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: please refer to Any other information on results incl. tables
test preparations were observed to be clear, colourless solutions throughout the duration of the test.
Results with reference substance (positive control):
- Results with reference substance valid? The results from the positive control with potassium dichromate were within the normal range for this reference material.
- Relevant effect levels: The No Observed Effect Concentrations after 24 and 48 hours were 1.0 mg/L and 0.56 mg/L respectively, based upon zero immobilisation at this concentration.
- Limit test: no
- Dose-response test: yes
- ECx: The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/L (sd = 0.24).
Reported statistics and error estimates:
Evaluation of data
The EC 50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used where two or more partial responses to exposure are shown.
The time-weighted mean measured test concentrations were calculated as follows:
TWM = (C0 - C1) / (ln(C0) - ln(C1))
where:
TWM = time-weighted mean measured test concentration (mg/L)
C0 = measured concentration at the start of the test (mg/L)
C1 = measured concentration at the end of the test (mg/L)

Evaluation of data
An estimate of the EC 50 value at 3 hours was given by inspection of the immobilisation data.
The EC 50 values and associated confidence limits at 24 and 48 hours was calculated using the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999).
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

Any other information on results incl. tables

Observations on test material solubility

The test preparations were observed to be clear, colourless solutions throughout the duration of the test.

 

Verification of test concentrations

As the test concentrations at the bottom of the test concentration range were close to the limit of quantitation (LOQ) of the analytical method, the 100 and 10 mg/L stock solutions were also analysed at 0 and 24 hours to indicate the test system had been dosed correctly.

Chemical analysis of the 100 and 10  mg/L stock solutions at 0  and 24 hours showed measured test concentrations of 93% to 112% of nominal indicating that the system had been dosed correctly.

Analysis of the fresh media (see Appendix 1) at 0 and 24 hours showed measured concentrations of 80% to 107% of nominal with the exception of 0.010, 0.018 and 0.032 mg/L at 24 hours which showed measured concentrations of 58%, 66% and 72% of nominal respectively.  Although these concentrations were outside the 80% to 120% acceptance limits the frozen duplicate samples were not analysed as these concentrations were below the No Observed Effect Concentration.

Analysis of the 24-Hour old media (see Appendix 1) at 24 and 48 hours showed measured test concentrations to range from less than the limit of quantitation (LOQ) to 57% of nominal.  These results were in line with the stability analyses which indicated that the test material was unstable under the test conditions employed.  

Given that a marked decline in measured test concentrations of the parent material was observed over each 24-Hour renewal period it was considered justifiable to present a ‘worst case’ analysis of  the data by calculating the results based on the time-weighted  mean  measured  test concentrations also.  

The time-weighted mean measured test concentrations were calculated as follows:

 Nominal Concentration (mg/L)

 Time-weighted Mean Measured Concentration (mg/L)

 Percentage of Nominal Concentration

 0.010

 0.0026

 26

 0.018

 0.0044

 25

 0.032

 0.0089

 28

 0.056

 0.020

 35

 0.10

 0.046

 46

 0.18

 0.089

 50

 0.32

 0.19

 60

 0.56

 0.35

 62

 1.0

 0.65

 65

Analysis of the immobilisation data by the maximum-likelihood probit method (Finney 1971) at 24 and 48 hours based on the time-weighted mean measured test concentrations gave the results indicated above (please refer to Effect concentrations).

The use of time-weighted mean measured concentrations was considered not to significantly alter the results of the test.

Positive Control

The results from the positive control with potassium dichromate were within the normal range for this reference material.  The mean 48-Hour EC 50  value calculated from all positive controls was 0.78 mg/l (sd = 0.24).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In a semi-static test according to OECD 202 with Daphnia magna the test substance showed a 48h-EC50 = 0.12 mg/L (TWA) and 48h-EC0: 0.02 mg/L (TWA).