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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:
13 January 2009 to 5 February 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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 relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 19 August 2008; Date of signature: 04 March 2009

Test material

Constituent 1
Details on test material:
Sponsor's identification : H-MI ammonium salt
Description : dark red solid
Purity : 90.3 wt%
Batch number : MB-1
Date received : 4 December 2008
Storage conditions : room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Theoretical concentration of 10 mg/l, nominal concentration of 100 mg/l .
- Sampling method: The test material concentration in the test samples was determined spectrophotometrically using an external standard.
- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test material was dissolved directly in reconstituted water.
- Controls: potassium dichromate

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: In-house laboratory cultures.
- Age at study initiation (mean and range, SD): <24 hours old
- Weight at study initiation (mean and range, SD): Not stated.
- Length at study initiation (length definition, mean, range and SD): Not stated.
- Feeding during test: yes
- Food type: Algae (Chlorella sp.)
- Frequency: Daily.

Study design

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

Test conditions

Hardness:
250 mg/l as CaC03
Test temperature:
Approximately 20°C recorded daily using a Hanna Instruments HI 93510 digital thermometer.
pH:
8.0 recorded daily using a WTW pH/Oxi 3401 pH and dissolved oxygen meter.
Dissolved oxygen:
8.6 - 9.1 mg O2/1 recorded daily using a WTW pH/Oxi 3401 pH and dissolved oxygen meter.
Salinity:
Not applicable, fresh water.
Nominal and measured concentrations:
Nominal concentrations: 100 mg/l.
Measured concentrations: 99% - 101% of nominal.
Details on test conditions:
TEST SYSTEM
- Test vessel
- Type: Covered.
- Material, size, headspace, fill volume: 250 ml glass jars containing approximately 200 ml of test preparation.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): Not applicable.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water
i) Stock Solutions
a) CaCI2.2H20 : 11.76 g/l
b) MgS04.7H20 : 4.93 g/l
c) NaHC03 : 2.59 g/l
d) KCI : 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 HCI and was aerated until the dissolved oxygen concentration was approximatelyair-saturation value.

- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
-light intensity: Approximately 500 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
An estimate of the ECso values was given by inspection of the immobilisation data.
The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and standard error of the response curves were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/l
- Results used to determine the conditions for the definitive study: Definitve test: 100 mg/l
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: None observed.
- Observations on body length and weight: No observations made.
- Other biological observations: Immobilisation.
- Mortality of control: The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/I (sd = 0.21).
- Other adverse effects control: None
- Abnormal responses: None observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test preparation was observed to be a clear red solution throughout the test. The control media was observed to be a clear, colourless solution throughout the test.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes, the No Observed Effect Concentration after 24 and 48 hours was 0.32 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration. The slopes and their standard errors of the response curves at 24 and 48 hours were 8.1 (SE = 1.7) and 8.6 (SE = 1.8) respectively. The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/I (sd = 0.21).
- Mortality: 100% immobilisation after 48 hours.
- EC50/LC50: EC50 > 3 hrs = > 3.2 mg/l; 24 hrs = 0.82 mg/l and 48 hours 0.71 mg/l.
Reported statistics and error estimates:
The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and standard error of the response curves 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.

Any other information on results incl. tables

Range-finding Test

No immobilisation was observed at the test concentrations of 0.10, 1.0, 10 and 100 mg/l. Based on this information, a single test concentration of four replicates, of 100 mg/l was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EEC Test Guidelines, no immobilisation or adverse reactions to exposure were observed.

Definitive Test

Immobilisation data

There was one immobilisation in 20 daphnids exposed to a test concentration of 100 mg/l for a period of 48 hours. This immobilisation was thought to be due to natural causes and not a true toxic effect. Inspection of the immobilisation data gave the following results:

Time (h)

EC50 (mg/l)

95% Confidence limits (mg/l)

24

>100

-

48

>100

-

The No Observed Effect Concentration after 24 and 48 hours exposure was 100 mg/l. The No Observed Effect Concentration is based upon no significant immobilisation at this concentration. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.

Verification of test concentrations

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 99% to 101% of nominal value and so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations only.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of greater than 100 mg/l. The No Observed Effect Concentration at 48 hours was 100 mg/l.
Executive summary:

Introduction. A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in 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, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a concentration of 100 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results. The 48-Hour EC50 for the test material to Daphnia magna based on nominal test concentrations was greater than 100 mg/l and correspondingly the No Observed Effect Concentration was 100 mg/l.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 99% to 101% of nominal value and so the results are based on nominal test concentrations only. The 48-Hour EC50 for the reference material to Daphnia magna based on nominal concentrations was 0.71 mg/l with 95% confidence limits of 0.61 - 0.81 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

Conclusion. The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of greater than 100 mg/l. The No Observed Effect Concentration at 48 hours was 100 mg/l.