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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:
14-30 April 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Recently conducted GLP compliant study using the most recent test methods

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): JKY-214
- Physical state: white solid
- Analytical purity: No information
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 2.0mg/l
- Sampling method: untreated in duplicate
- Sample storage conditions before analysis: c. -20C

Test solutions

Vehicle:
yes
Details on test solutions:
Media preparation trial

Information provided by the manufacturer indicated that the test material was insoluble in water. Pre-study solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. A test concentration of 2.0 mg/l (by visual inspection) was obtained using a preliminary solution in dimethylformamide.
Based on this information the test material was categorised as being a 'difficult substance' as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.

Saturated solution preparation

An amount of test material (1125 mg) was dispersed, in duplicate in 22.5 litres of reconstituted water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21 °C for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
- Centrifugation at 10000 g for 30 minutes
- Centrifugation at 40000 g for 30 minutes
- Filtration through a 0.2 urn Sartorius Sartopore filter (approximate 1 litre discarded in order to pre-condition the filter)
- Filtration through a 0.2 urn Sartorius Sartopore filter (approximate 2 litres discarded in order to pre-condition the filter)

Solvent spike preparation

An amount of test material (200 mg) was dissolved in dimethylformamide and the volume adjusted to 10 ml to give a 200 mg/10 ml solvent stock solution. An aliquot (200 ul) of this 200 mg/10 ml solvent stock solution was dispersed, in triplicate, in 2 litres of reconstituted water with the aid of magnetic stirring for approximately 10 minutes, 24 hours or 48 hours prior to taking samples for chemical analysis after the following pre-treatments:
- Untreated
- Centrifugation at 10000 g for 30 minutes
- Centrifugation at 40000 g for 30 minutes
- Filtration through a 0.2 urn Gelman Acrocap filter (approximate 100 ml discarded in order to pre-condition the filter)
- Filtration through a 0.2 pm Gelman Acrocap filter (approximate 500 ml discarded in order to pre-condition the filter)

Range-finding test

Based on the results obtained from the pre-study media preparation trial, the test material was prepared using a solvent spike method of preparation prior to removal of any undissolved test material by centrifugation at 40000 g for 30 minutes to give a nominal test concentration of 0.20 mg/l.

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 0.0020, 0.020 and 0.20 mg/l. The test material was prepared using a preliminary solution in dimethylformamide.

An amount of test material (200 mg) was dissolved in dimethylformamide and the volume adjusted to 10 ml to give a 200 mg/10 ml solvent stock solution. An aliquot (100 pi) of this solvent stock solution was dispersed in 1 litre of reconstituted water to give a 2.0 mg/l stock dispersion. Undissolved test material was then removed by centrifugation at 40000 g for 30 minutes to give a 0.20 mg/l test concentration. Serial dilutions were prepared in reconstituted water from the 0.20 mg/l test concentration to give the remainder of the test concentrations of 0.020 and 0.0020 mg/l.

Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive test

Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 0.20 mg/l to confirm that at the highest attainable test concentration of 0.20 mg/l, no immobilisation or adverse reactions to exposure were observed.

Chemical analysis of the test preparations showed the measured concentration of the fresh media to be lower than the expected nominal. In addition, analysis of the 48-Hour old media showed a decline in measured concentration. Given this the results were based on the geometric mean measured concentration which was calculated to be 0.034 mg/l.

Experimental preparation

For the purpose of the definitive test the test material was prepared using a preliminary solution in dimethylformamide.

An amount of test material (200 mg) was dissolved in dimethylformamide and the volume adjusted to 10 ml to give a 200 mg/10 ml solvent stock solution. An aliquot (200 ul) of this solvent stock solution was dispersed in 2 litres of reconstituted water to give a 2.0 mg/l stock dispersion. Undissolved test material was then removed by centrifugation at 40000 g for 30 minutes to give the nominal test concentration of 0.20 mg/l (0.034 mg/l based on geometric mean measured concentration).

Each stock solution/dispersion 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.

Verification of test concentrations

Water samples were taken from the solvent control (replicates Rt - R4 pooled) and the 0.20 mg/l test group (replicates Ri - 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 organisms

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

Adult Daphnia Magna 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:
None

Test conditions

Hardness:
Approximately 250 mg/I as CaCO3
Test temperature:
21°C to 22°C
pH:
7.8 to 8.1
Dissolved oxygen:
7.5 to 8.9 mg O2/l
Salinity:
Not applicable.
Nominal and measured concentrations:
0.20 mg/I (nominal value)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml glass jars
- Type (delete if not applicable): Covered to reduce evaporation
- Material, size, headspace, fill volume: 250 ml
- Aeration: None
- 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/test vessel
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: Not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water
- Total organic carbon: Not measured
- Particulate matter: Not measured
- Metals: Not measured
- Pesticides: Not measured
- Chlorine: Not measured
- Alkalinity: Not measured
- Ca/mg ratio: Not measured
- Conductivity: Not measured
- Culture medium different from test medium: Reconstituted water
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: Sixteen hours of continuous artificial light and eight hours continuous darkness with 20 minute dawn and dusk transition periods.
- Light intensity: approximately 800 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 24 h and 48 h EC50 values

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Factors of10.
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: 0.0020, 0.020 and 0.20 mg/l. The test material was prepared using a preliminary solution in dimethylformamide.

- Results used to determine the conditions for the definitive study: "Limit test" was conducted at a concentration of 0.20 mg/l to confirm that at the highest attainable test concentration of 0.20 mg/l, 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.:
> 0.034 mg/L
Nominal / measured:
nominal
Conc. based on:
dissolved
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
> 0.034 mg/L
Nominal / measured:
nominal
Conc. based on:
dissolved
Basis for effect:
mobility
Details on results:
Pre-study Media Preparation Trial

The results of the media preparation trial indicated that the test material adsorbed to the filter matrix. Analysis of the solvent spike samples indicated that centrifugation at 10000 g for 30 minutes may not remove all the undissolved test material. The results from the saturated solution samples following centrifugation were variable indicating that centrifugation may not remove the greater excess of test material present using this method of preparation.

The results from the solvent spike method of preparation did not show an increase in measured concentration with prolonged stirring.

It was therefore considered that the most appropriate method of preparation for the test material was as a solvent spike at an initial 2.0 mg/l. Undissolved test material was then removed by centrifugation at 40000 g for 30 minutes to give a nominal test concentration of 0.20 mg/l.

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 (see below).

No immobilisation was observed at the test concentrations of 0.0020, 0.020 and 0.20 mg/l.

Based on this information, a single test concentration of four replicates, of 0.20 mg/l was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the highest attainable test concentration of 0.20 mg/l, no immobilisation or adverse reactions to exposure were observed.

Chemical analysis of the test preparations showed the measured concentration of the fresh media to be lower than the expected nominal. In addition, analysis of the 48-Hour old media showed a decline in measured concentration. Given this the results were based on the geometric mean measured concentration which was calculated to be 0.034 mg/l.

Definitive Test

Verification of test concentrations

Analysis of the freshly prepared test concentrations at 0 hours showed measured concentrations of 0.0654 and 0.0641 mg/l. These concentrations were lower than the expected 0.20 mg/l observed from the media preparation trial. A Study to Determine the General Physico-Chemical Properties of the test material (Harlan Laboratories Ltd Project Number: 0696/0104) showed the water solubility of the test material to be variable with values determined at less than 2 x 10-6 to 2.7 x 10-5 g/l. It was therefore considered that the differences observed in measured concentration between the media preparation trial and the test was due to the poor water solubility of the test material.

Analysis of the old media at 48 hours showed measured concentrations of 0.0192 and 0.0167 mg/l.

This decline in measured concentration over the 48-Hour test period was contrary to the stability analyses which showed the test material to be stable in the test medium over 48 hours. A Study to Determine the General Physico-Chemical Properties of the test material (Harlan Laboratories Ltd Project Number: 0696/0104) gave a partition coefficient, log Pow of 3.37 and an adsorption coefficient, log Koc of 5.34 thereby indicating a high potential for the test material to adsorb to organic matter and/or bioaccumulate in the test organisms

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The geometric mean measured concentration was calculated to be 0.034 mg/l.

Immobilisation data

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

There was no significant immobilisation in 20 daphnids exposed to a geometric mean measured test concentration of 0.034 mg/l for a period of 48 hours. Inspection of the immobilisation data gave the as 24 and 48 Hr EC50 at > 0.034.

The No Observed Effect Concentration after 24 and 48 hours exposure was 0.034 mg/l. The No Observed Effect Concentration is based upon no significant immobilisation at this concentration.

A single daphnid was observed immobilised in both the control and 0.034 mg/l test concentration. These were not considered to be significant given that less than 10% immobilisation occurred and were considered to be possibly due to natural causes.

Observations on test material solubility

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

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.

A single temperature was observed to be slightly in excess of the range given in the protocol of 20 +/- 1°C. This deviation was considered not to have affected the outcome or the validity of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and the temperature range was within guideline specification.
Results with reference substance (positive control):
Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the probit method (Finney 1971) at 48 hours based on the nominal test concentrations gave the following results:

3h EC50 >3.2 mg/l
24h EC50 0.82 mg/l; 95% Confidence limits 0.71 - 0.94
48h EC50 0.71 mg/l ; 95% Confidence limits 0.62 - 0.81
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/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 ImmobilisedDaphnia{Initial Population: 10 Per Replicate)

 

 

24 Hours

48 Hours

Control

0

0

Solvent Control

0

0

0.0020

0

0

0.020

0

0

0.20

0

0

 

Table 2 - Cumulative Immobilisation Data in the Definitive Test

 

Geometric Mean Measured 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

 

 

 

R2

0

 

 

1

 

 

 

 

 

0

0

 

1*

5

 

R3

0

 

 

0

 

 

 

R4

0

 

 

0

 

 

Solvent Control

R1

0

 

 

0

 

 

 

R2

0

 

 

0

 

 

 

 

 

0

0

 

0

0

 

R3

0

 

 

0

 

 

 

R4

0

 

 

0

 

 

0.034

R1

0

 

 

0

 

 

 

R2

0

 

 

1

 

 

 

 

 

0

0

 

1*

5

 

R3

0

 

 

0

 

 

 

R4

0

 

 

0

 

 

 

* Considered to be possibly due to natural causes given that <10% immobilisation observed

Table 3 - Cumulative Immobilisation Data in the Positive Control

 

Nominal Concentration (mg/l)

Cumulative ImmobilisedDaphnia(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

1

1

2

10

2

2

4

20

1.0

0

0

0

0

7

8

15

75

9

9

18

90

18

0

0

0

0

10

10

20

100

10

10

20

100

3.2

0

0

0

0

10

10

20

100

10

10

20

100

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 based on geometric mean measured concentrations of greater than 0.034 mg/l. Correspondingly the No Observed Effect Concentration was 0.034 mg/l. This was the determined limit of solubility on the test media. The substance is not considered acutely toxic to Daphnia at the highest attainable concentration.
Executive summary:

The acute toxicity to Daphnia has been assessed by means of exposure to Daphnia magna according to EU method C2in compliance with GLP. Due to the extremely low water solubility the maximum attainable concentration was 0.034 mg/l. As no mortality or clinical signs were observed at the highest attainable test concentration the substance is considered not acutely toxic to Daphnia.