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Diss Factsheets

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:
The range-finding test was conducted between 28 March 2007 and 30 March 2007 and the definitive test between 1 May 2007 and 3 May 2007.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification: C-Y9
Description : yellow powder
Lot Number : MB-1
Date received : 6 March 2007
Storage conditions: room temperature in the dark over silica gel

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
100 mg/l.

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

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/l. The test material was dissolved directly in water.

An amount (100 mg) of test material was dissolved directly in reconstituted water and the volume adjusted to 1 litre to give the 100 mg/l test concentration. Serial dilutions of the 100 mg/l test concentration were made to give the test concentrations 10, 1.0 and 0.10 mg/l.
Each stock solution and 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 approximately 20°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. Each 250 ml test and control vessel contained 200 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 material.

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

Experimental preparation:
For the purpose of the definitive test the test material was dissolved directly in reconstituted water.

An amount (100 mg) of test material was dissolved directly in reconstituted water and the volume adjusted to 1 litre to give the 100 mg/l test concentration.

The 100 mg/l test concentration was inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 48 hours .





















Test organisms

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

Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water 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 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.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Test conditions

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 test vessels were maintained in a temperature controlled room at 21°C to 22°C.
pH:
pH was recorded at the start and termination of the test.

pH at start and termination of test: 8.0
Dissolved oxygen:
Dissolved oxygen concentrations were recorded at the start and termination of the test.
The test water had a dissolved oxygen content of greater than or equal to 8.4 mg O2/l.
Salinity:
Not measured.
Nominal and measured concentrations:
Nominal concentration: 100 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml glass jars.
- Type: The test vessels were covered to reduce evaporation.
- Material, size, headspace, fill volume: 250 ml glass jars containing 200 ml of test preparation.
- Aeration: The test vessels were not aerated.
- Renewal rate of test solution (frequency/flow rate): The test preparations were not renewed during the exposure period.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4 (The control group was maintained under identical conditions but not exposed to the test material).


TEST MEDIUM / WATER PARAMETERS
The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

The reconstituted water 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.

OTHER TEST CONDITIONS
- Photoperiod: The test vessels were maintained 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.


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.

TEST CONCENTRATIONS
- 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.
- Results used to determine the conditions for the definitive study:
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg/l to confirm that at the maximum concentration given in the OECD/EEC Test Guidelines, no immobilisation or adverse reactions to exposure were observed.
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:
Range-finding Test:
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in Table 1.

No immobilisation was observed at the test concentrations of 0.10, 1.0, and 10 mg/l. However, a single immobilised daphnid at 100 mg/l after 48 hours was observed. This was considered to be due to natural causes as no other immobilisation was observed and this single immobilisation amounted to 10% which is within the limits allowed for immobilisation in the Test Guidelines.

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:

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2.

There was no immobilisation in 20 daphnids exposed to a test concentration of 100 mg/l for a period of 48 hours.

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 material solubility:
Throughout the duration of the test the test concentrations were observed to be clear, yellow solutions.

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 or pH.

Verification of test concentrations:
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal value and so it was considered justifiable to estimate the EC50 values in terms of the nominal test concentrations only.








Results with reference substance (positive control):
Positive Control:
Cumulative immobilisation data from the exposure of Daphnia magna to the reference material during the positive control are given in Table 3.

Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the probit method (Finney 1971) at 24 hours and the geometric mean method at 48 hours based on the nominal test concentrations gave the following results:

Time (h) EC50 (mg/l) 95% confidence limits (mg/l)
3 >3.2 -
24 1.2 1.1 - 1.4
48 0.75 0.56 - 1.0*

*Concentrations resulting in 0% and 100% immobilisation respectively.

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 slope and standard error of the response curve at 24 hours was 6.9 (SE = 1.4). Due to the unsuitable nature of the data it was not possible to calculate the slope and standard error of the response curve at 48 hours.

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 0.81 mg/l (sd = 0.21).



Any other information on results incl. tables

Table 1               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

1[*]


[*]Immobilisation considered to be due natural causes as no other immobilisation was observed.

 

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

Table 3               Cumulative Immobilisation Data in the Positive Control

Nominal
Concentration
(mg/l)

Cumulative Immobilised Daphnia
(Initial Population: 10 Per Replicate)

3 Hours

24 Hours

48 Hours

R1

R2

Total

%

R1

R2

Total

%

R1

R2

Total

%

Control

0

0

0

0

0

0

0

0

0

0

0

0

0.32

0

0

0

0

0

0

0

0

0

0

0

0

0.56

0

0

0

0

0

0

0

0

0

0

0

0

1.0

0

0

0

0

2

4

6

30

10

10

20

100

1.8

0

0

0

0

8

9

17

85

10

10

20

100

3.2

0

0

0

0

10

10

20

100

10

10

20

100


R1– R2= Replicates 1 and 2

Physico-Chemical Measurements

Nominal
Concentration
(mg/l)

0 Hours

24 Hours

48 Hours

pH

mg O2/1

%*

T°C

TºC

pH

mg O2/1

%*

T°C

Control

R1

8.0

8.6

99

22

21

8.0

8.7

98

21

 

R2

8.0

8.4

97

22

21

8.0

8.7

98

21

 

R3

8.0

8.4

97

22

21

8.0

8.7

98

21

 

R4

8.0

8.6

99

22

21

8.0

8.8

99

21

100

 

R1

8.0

8.5

98

22

21

8.0

8.7

98

21

R2

8.0

8.6

99

22

22

8.0

8.8

99

21

R3

8.0

8.5

98

22

21

8.0

8.7

98

21

R4

8.0

8.5

98

22

21

8.0

8.7

98

21

 


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

R1– R4= Replicates 1 to 4

Verification of Test Concentrations

Results:

Sample

Nominal

Concentration

(mg/l)

Concentration

Found (mg/l)

Expressed as a Percent of the Nominal Concentration (%)

0 Hours

Control

<LOQ

-

 

100 R1-R2

103

103

 

100 R3-R4

98.6

99

48 Hours

Control

<LOQ

-

 

100 R1-R2

101

101

 

100 R3-R4

98.9

99


LOQ = Limit of quantitation

R1– R4= Replicates 1 to 4

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 of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration 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 Directive 92/69/(which constitutes Annex V of Council Directive 67/548/).

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 21°C to 22°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 21°C to 22°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results.

The 48-Hour EC50for 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 be near nominal and so the results are based on nominal test concentrations only.

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

The 48-Hour EC50for the reference material to Daphnia magna based on nominal concentrations was 0.75 mg/l with 95% confidence limits of 0.56 – 1.0 mg/l. The No Observed Effect Concentration was 0.56 mg/l.