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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 13 June 2011 and 15 July 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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.
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)
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
In the definitive test a single concentration of 100 mg ai/l was tested.

- Sampling method:
Water samples were taken from the control (replicates R1 - R4 pooled) and the 100 mg ai/I test group (replicates R1 - R2 and R3 - R4 pooled) at 0 and 48 hours for quantitative analysis.

- Sample storage conditions before analysis:
The 0 and 24 hour samples were stored at approximately -20°C prior to analysis.
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Range-finding test:
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.
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 active ingredient (ai)/l. The test item was dissolved directly in reconstituted water. At the request of the Sponsor the test concentrations were corrected for a water content of 4.3%.
An amount of test item (104 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give the 100 mg ai/l test concentration from which serial dilutions were prepared, in reconstituted water, to give the remainder of the test series of 10, 1.0, and 0.10 mg ai/l.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 21°C to 22°C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Some of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed throughout the duration of the test and that the temperatures were within the test guideline specification. Each 250 ml test and control vessel contained 250 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna 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 at approximately -20°prior to analysis.

Definitive test:
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg ai/l to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no immobilisation or adverse reactions to exposure were observed.

Experimental preparation:
For the purpose of the definitive test the test item was dissolved directly in reconstituted water. At the request of the Sponsor the test concentrations were corrected for a water content of 4.3%.
An amount of test item (209 mg) was dissolved in reconstituted water and the volume adjusted to 2 litres to give the 100 mg ai/l test concentration.
The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration of the test item in the test preparations was verified by chemical analysis at 0 and 48 hours.
Test organisms (species):
Daphnia magna
Details on test organisms:
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 21°C to 22°C. A temperature at which the stock daphnids were kept was observed to be slightly in excess of the range given in the study plan of 20 ± 1°C. This deviation was considered not to have affected the outcome or the validity of the test as there was no effect on survival and reproduction of the stock cultures and the temperature range was within guideline specification. 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 type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
Not applicable.
Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.
Test temperature:
Water temperature was recorded daily throughout the test. The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
The results of the physico-chemical measurements are given in the results section. Temperature was maintained at 21°C to 22°C throughout the test. While there were no treatment related differences for oxygen concentration, differences in pH were observed between the control and 100 mg ai/l test concentration throughout the test.
Some of the temperatures were measured to be slightly in excess of the 20 ± 1C given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed throughout the duration of the test and that the temperatures were within the test guideline specification.
pH:
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi Handheld meter.
The results of the physico-chemical measurements are given in the results section.
Dissolved oxygen:
Dissolved oxygen concentrations were recorded at the start and termination of the test.
The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.
Salinity:
freshwater used.
Nominal and measured concentrations:
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg/L (nominal) to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no immobilisation or adverse reactions to exposure were observed.
Analysis of the test preparations at aand 48 hours showed measured test concentrations to range from 96% to 99% of nominal value and so it was considered justifiable to estimate the ECso values in terms, of the nominal test concentrations only.
Details on test conditions:
Exposure conditions:
In the definitive test 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21°C to 22°C with a photoperiod of 16 hours light 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 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.


Test Water:
Reconstituted water was used for both the range-finding and definitive test and is defined below:
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.









Reference substance (positive control):
yes
Remarks:
Potassium dichromate
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: 95% CL not stated
Details on results:
Range-finding Test:
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 (see any other information on results incl. tables section).
No immobilisation was observed at the test concentrations employed in the test.
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/EC Test Guidelines, no immobilisation or adverse reactions to exposure were observed.
Chemical analysis of the 100 mg/L test preparation at 0 and 48 hours (see Appendix 5 - attached background material) showed measured concentrations of 93% and 85% of nominal value indicating that the test item was stable under test conditions.

Definitive Test:
Immobilisation data:
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2 (see any other information on results incl. tables section).
There was no immobilisation in 20 daphnids exposed to a test concentration of
100 mg ai/l for a period of 48 hours. Inspection of the immobilisation data gave the following results:
Time (h) EC50 (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 zero immobilisation at this concentration.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Observations on test item solubility:
The test preparations were observed to be clear, colourless solutions throughout the duration of the test.

Verification of test concentrations:
Analysis of the test preparations at 0 and 48 hours (see Appendix 5 - attached background material) showed measured test concentrations to range from 96% to 99% of nominal value and so it was considered justifiable to estimate the EC50 values in terms of the nominal test concentrations only.

Physico-chemical measurements:
Temperature was maintained at 21°C to 22°C throughout the test. While there were no treatment related differences for oxygen concentration, differences in pH were observed between the control and 100 mg/L test concentration throughout the test.

Validation Criteria
The results of the test are considered valid given that:
-Zero immobilisation or other signs of disease or stress was observed in the control daphnids.
-The dissolved oxygen concentration at the end of the test does was 100% Air Saturation Value (ASV) in the control vessels.



Results with reference substance (positive control):
Positive Control
A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilisation data by the maximum-likelihood probit method (Finney 1971 ) at 24 and 48 hours based on the nominal test concentrations gave the following results:

24 h EC50: 1.5 mg/l (95% CL 1.3 - 1.8 mg/l)
48 h EC50: 0.99 mg/l (95% CL 0.85 - 1.1mg/l)

The No Observed Effect Concentration after 24 and 48 hours was 0.56 mg/l. The No Observed Effect Concentration 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.


Table1              Cumulative Immobilisation Data in the Range-finding Test

Nominal
Concentration
(mg/L)

Cumulative Immobilised Daphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

0.10

0

0

1.0

0

0

10

0

0

100

0

0

Table 2              Cumulative Immobilisation Data in the Definitive Test

Nominal
Concentration
(mg/L)

Cumulative Immobilised Daphnia
(Initial Population: 5 Per Replicate)

24 Hours

48 Hours

No. Per

Replicate

Total

%

No. Per

Replicate

Total

%

Control

R1

0

0

0

0

0

0

 

R2

0

0

 

R3

0

0

 

R4

0

0

100

R1

0

0

0

0

0

0

 

R2

0

0

 

R3

0

0

 

R4

0

0

R1– R4= Replicates 1 to 4

Physico-Chemical Measurements

Nominal
Concentration
(mg/L)

0 Hours

24 Hours

48 Hours

pH

mg O2/l

%*

T°C

TºC

pH

mg O2/l

%*

T°C

Control

R1

8.1

9.1

102

21

22

7.7

8.7

100

22

 

R2

7.9

9.1

102

21

22

7.7

8.7

100

22

 

R3

7.8

9.1

102

21

22

7.8

8.7

100

22

 

R4

7.8

9.1

102

21

22

7.8

8.7

100

22

100

 

R1

6.8

9.1

102

21

22

7.6

8.7

100

22

R2

6.7

9.1

102

21

22

7.5

8.7

100

22

R3

6.6

9.1

102

21

22

7.4

8.7

100

22

R4

6.5

9.1

102

21

22

7.4

8.6

99

22

*ASV= Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

R1– R4= Replicates 1 to 4

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.
Executive 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, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a concentration of 100 mg/L for 48 hours at a temperature of 21°C to 22°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours. At the request of the Sponsor all concentrations were corrected for a test item water content of 4.3%.

Results

The 48-Hour EC50 for the test item to Daphnia magna based on nominal test concentrations was greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 96% to 99% of nominal value and so the results are based on nominal test concentrations only.

 

Description of key information

In a single key study conducted in accordance with OECD 202, no effects were seen in the freshwater invertebrate species Daphnia magna in a limit test conducted at 100 mg/L.

Key value for chemical safety assessment

Additional information

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.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a concentration of 100 mg/L for 48 hours at a temperature of 21°C to 22°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

The 48-Hour EC50 for the test item to Daphnia magna based on nominal test concentrations was greater than 100 mg/L.. 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 96% to 99% of nominal value and so the results are based on nominal test concentrations only.

As there were no effects seen in Daphnia magna up to the regulatory limit concentration of 100 mg/L, no key effect value for chemical safety assessment is relevant for fish and the substance is not considered to be hazardous to this aquatic trophic level.