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:
Experimental Starting Date: 05 June 2014 Experimental Completion Date: 24 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Remarks:
There were no deviations (unplanned changes) from the study plan.
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Remarks:
There were no deviations (unplanned changes) from the study plan.
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. The test item was initially dissolved in dimethylformamide at a concentration of 100 mg/10 mL. An aliquot of this solvent stock solution was then dispersed in an appropriate volume of test media with the aid of magnetic stirring for approximately
10 minutes prior to removing any undissolved test item present by filtration (0.2 µm Gelman Acrocap, first approximate 500 mL discarded in order to pre-condition the filter) to give a nominal concentration of 1.0 mg/L.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: BMS-587172-01
- Substance type: Chemical intermediate
- Physical state: White powder
- Analytical purity: 99.9%
- Lot/batch No.: 2G71626N
- Expiration date of the lot/batch: 27 August 2014
- Storage condition of test material:Room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
The analytical methodology was not validated prior to the analysis of range-finding samples; full validation was later conducted specifically for the test concentration range employed in the definitive test. The following nominal test concentrations were assigned to the definitive test: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L.

- Sampling method:
Water samples were taken from the solvent control and the each test group at 0 and 24 hours (fresh media from the bulk test preparation) and at 24 and 48 hours (old media from replicates R1- R4 pooled) for quantitative analysis. Samples were stored frozen prior to analysis.
 
- Sample storage conditions before analysis:
Samples used immediately.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:
Experimental Preparation:
Prior to use of the test item its appearance was checked against its documented description as a confirmation check used to verify its identity.

A nominal amount of test item (100 mg) was dissolved in dimethylformaide and the volume adjusted to 10 mL to give the 100 mg/10 mL solvent stock solution. An aliquot (500 µL) of the solvent stock solution was added to 5 liters of test water and stirred using a magnetic stirrer for 10 minutes. After stirring, any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to give a 1.0 mg/L test concentration. Aliquots (20, 64, 200 and 640 mL) of the 1.0 mg/L test concentration were then each separately added to a final volume of 2 liters of test water to give the remaining test concentrations of 0.010, 0.032, 0.10 and 0.32 mg/L.

All preparation was carried out under laboratory safety lighting due to the light sensitive nature of the test item.

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

The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0 and 24 hours (fresh media) and 24 and 48 hours (old media).

- Controls:
A positive control (Harlan Study Number 41400711) used potassium dichromate as the reference item. The positive control was conducted between 05 March 2014 and 07 March 2014.


Test organisms

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 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:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Any immobilization 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 immobilized if they were unable to swim within 15 seconds after gentle agitation.

Test conditions

Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.
Test temperature:
The water temperature was recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24 hour old test preparations. The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

Water temperature was maintained at approximately 20 °C throughout the test,
pH:
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
The pH was measured using a WTW pH/Oxi 340I pH meter.
There were no treatment related differences for pH.
Dissolved oxygen:
The dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at
24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24 hour old test preparations. The dissolved oxygen concentration was measured using a Hach HQ30d Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

There were no treatment related differences for dissolved oxygen concentration.
Salinity:
Not applicable as a fresh water study was conducted
Nominal and measured concentrations:
Range finding study - nominal concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.0010, 0.010, 0.10 and 1.0 mg/L.

Definitive study - The nominal and time weighted mean measured test concentrations can be found below:
Nominal Test Concentration (mg/L) Time-Weighted Mean Measured Test Concentrations (mg/L)
0.032 0.0073
0.10 0.017
0.32 0.059
1.0 0.22
Details on test conditions:
TEST SYSTEM
- Test vessel:
As in the range-finding 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, control and solvent control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 20 °C in darkness. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control and solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100 µL/L of dimethylformamide.

Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. 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.

- Source/preparation of dilution water:
Reconstituted water (ISO medium) was used for both the range-finding and definitive test is defined below:

Ingredient Final Concentration (mg/L)
CaCl2.2H2O 294
MgSO4.7H2O 123
NaHCO3 64.75
KCl 5.75

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.

- Culture medium different from test medium:
Reconstituted water (ISO medium) was used for both the range-finding and definitive test. The adult Daphnia were maintained in Elendt M7 medium prior to the study.

- Intervals of water quality measurement:
Not recorded

OTHER TEST CONDITIONS
- Photoperiod:
a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.

- Light intensity:
Not recorded

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:

The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.0010, 0.010, 0.10 and 1.0 mg/L.

Based on the results of the range-finding test the following nominal test concentrations were assigned to the definitive test: 0.010, 0.032, 0.10, 0.32 and 1.0 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.:
0.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% CL of 0.12 - 0.32 mg/l.
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.007 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
0.017 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Details on results:
Range-finding Test:
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1*.

No immobilization was observed at the test concentrations of 0.0010, 0.010 and 0.10 mg/L. However, immobilization was observed at 1.0 mg/L and sub lethal effects of slower movement and trailing debris were observed at 0.10 and 1.0 mg/L.

Based on this information test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L were selected for the definitive test.

Chemical analysis of the 0.010, 0.10 and 1.0 mg/L test preparations at 0 hours showed that measured concentrations of between less than the limit of quantification (LOQ, determined to be 0.0029 mg/L) and 0.37 mg/L were obtained, whilst at 48 hours measured concentrations of between less than the LOQ and 0.20 mg/L were obtained indicating that the test item was possibly unstable under test conditions.

* All tables can be found in the any other information on results section (including tables)

Definitive Test:
Verification of Test Concentrations:
Analysis of the freshly prepared test media at 0 and 24 hours showed measured test concentrations to range from 0.00620 to 0.351 mg/L. A decline in measured test concentration was observed in the old or expired test media at 24 and 48 hours to between 0.00523 and 0.164 mg/L and hence it was considered appropriate to calculate the results based on the time-weighted mean measured test concentration only in order to give a “worst case” analysis of the data.

A measured concentration of test item was obtained in the 24 hour old solvent control sample, however, analysis of the duplicate sample showed that a measured concentration of less than the limit of quantification was obtained suggesting that the original result was anomalous. This was considered not to have had an impact on the integrity of the study. In the 24 hour old 0.032 mg/L test sample, a measured test concentration of 37% of nominal was observed, which was an increase in test item concentration when compared to the 0 hour fresh result of 23% of nominal value, which, given the decline in measured concentration seen in every other test concentration, was considered to be anomalous. Analysis of the duplicate sample showed that a measured concentration of 28% of nominal value was obtained, therefore, the mean of the original and duplicate sample results was taken and used for calculation purposes.

The time-weighted mean measured test concentrations were determined to be:
Nominal Test Concentration (mg/L) Time-Weighted Mean Measured Test Concentrations (mg/L)
0.032 0.0073
0.10 0.017
0.32 0.059
1.0 0.22

Immobilization Data:
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2*.

Analysis of the immobilization data by the trimmed Spearman-Karber method (Hamilton et al, 1977) at 48 hours and inspection of the immobilization data at 24 hours gave the following results:

Time (h) EC50 (mg/L) 95% Confidence limits (mg/L)
24 >0.22 -
48 0.20 0.12 – 0.32

The No Observed Effect Concentrations after 24 and 48 hours exposure were 0.22 and 0.0073 mg/L respectively. The Lowest Observed Effect Concentrations after 24 and 48 hours exposure were greater than 0.22 and 0.017 mg/L respectively

* All tables can be found in the any other information on results section (including tables)

Observations on Test Item Solubility:
Throughout the duration of the test, the test preparations were observed to be clear colorless solutions.

Results with reference substance (positive control):
A positive control (Harlan Study Number 41400711) 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 immobilization data by the maximum-likelihood probit method (Finney, 1971*) at 24 hours and by the trimmed Spearman-Karber method (Hamilton et al 1977 *) at 48 hours based on the nominal test concentrations using the ToxCalc software package (ToxCalc 1999) gave the following results:

Time Point (Hours) EC50 (mg/L) 95% Confidence Limits (mg/L) (NOEC) (mg/L) (LOEC) (mg/L)
24 0.87 0.75 - 1.0 0.56 1.0
48 0.71 0.65 - 0.78 0.56 1.0

The No Observed Effect Concentration is based upon less than 10% immobilization at this concentration.

The results from the positive control with potassium dichromate were within the normal range for this reference item.
Reported statistics and error estimates:
Statistical Analysis:
The EC50 value and associated confidence limits at 48 hours was calculated by 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.

An estimate of the EC50 value at 24 hours was given by inspection of the immobilization data.

Any other information on results incl. tables

This section includes tables 1 and 2 which provides details on the immobilisation data within the range finding and definitive studies, and tables from the analytical investigations.

Table 1     Cumulative Immobilization Data in the Range-finding Test

Nominal
Concentration
(mg/L)

Cumulative Immobilized Daphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

Solvent Control

0

0

0.0010

0

0

0.010

0

0

0.10

0

0*

1.0

0

6**


*5 out of the 10 daphnids observed to be slower moving

**2 out of the remaining 4 daphnids observed to be trailing debris.

Table 2     Cumulative Immobilization Data in the Definitive Test

Nominal Concentration
(mg/L)

Time-Weighted Mean Measured Test

Concentration
(mg/L)

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

24 Hours

48 Hours

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Total

%

Control

-

0

0

0

0

0

0

0

0

0

0

0

0

Solvent Control

-

0

0

0

0

0

0

0

0

0

0

0

0

0.010

-

0

0

0

0

0

0

0

0

0

0

0

0

0.032

0.0073

0

0

0

0

0

0

0

0

0

0

0

0

0.10

0.017

0

0

0

0

0

0

0*

0*

0

0*

0

0

0.32

0.059

0

0

0

0

0

0

0**

0*

0

0*

0

0

1.0

0.22

0

0

0

0

0

0

2***

4

2§

3§§

11

55

R1– R4= Replicates 1 to 4

*1 out of the 5 daphnids observed to be slower moving

**2 out of the 5 daphnids observed to be slower moving

***2 out of the remaining 3 daphnids observed to be slower moving

§all of the remaining daphnids observed to be slower moving

§§1 out of the remaining 2 daphnids observed to be slower moving

Tables from the Analytical investigation:

Results for Spiked Recovery Samples:

Nominal Concentra­tion of
Test Item


cnom

Fortified Concentra­tion of Test Item in the Spiked Sample


cfort

Measured Concentra­tion of Test Item in the Sample Vial

x

Sample Preparation Factor



F

Determined Concentra­tion of Test Item in the Spiked Sample


c

Mean Analytical Recovery
Rate



R

Precision (Relative Standard Deviation of Recovery)

[mg/L]

[mg/L]

[mg/L]

 

[mg/L]

[%]

[%]

0.0100

0.0107

0.00278

4

0.0111

99

6.7

 

0.0107

0.00253

4

0.0101

 

0.0107

0.00288

4

0.0115

 

0.0107

0.00244

4

0.00977

 

0.0107

0.00263

4

0.0105

0.0500

0.0535

0.0128

4

0.0511

92

2.4

 

0.0535

0.0122

4

0.0488

 

0.0535

0.0121

4

0.0485

 

0.0535

0.0121

4

0.0485

 

0.0535

0.0121

4

0.0483

 Acceptance Target

 

 

 

80-120

<10

Results for Test Samples:

Time Point (hours)

Nominal Concentration of
Test Item in Test Sample

cnom [mg/L]

Measured Concentration
 of Test Item in Sample Vial

x

[mg/L]

Sample Preparation Factor

F

Determined Concentration of Test Item in Test Sample


c

[mg/L]

% of Nominal Concentration

[%]

0

Solvent Control

<LOQ

4

<LOQ

-

(Fresh Media)

0.032

0.00186

4

0.00742

23

 

0.10

0.00600

4

0.0240

24

 

0.32

0.0182

4

0.0728

23

 

1.0

0.0878

4

0.351

35

24

Solvent Control

0.00221 / <LOQ*

4

0.00883 / <LOQ*

- / -*

(Old Media)

0.032

0.00298 / 0.00227*

4

0.0119 / 0.00908*

37 / 28*

 

0.10

0.00523

4

0.0209

21

 

0.32

0.0141

4

0.0564

18

 

1.0

0.0410

4

0.164

16

24

Solvent Control

<LOQ

4

<LOQ

-

(Fresh Media)

0.032

0.00155

4

0.00620

19

 

0.10

0.00388

4

0.0155

16

 

0.32

0.0204

4

0.0815

25

 

1.0

0.0645

4

0.258

26

48

Solvent Control

<LOQ

4

<LOQ

-

(Old Media)

0.032

0.00131

4

0.00523

16

 

0.10

0.00248

4

0.00992

10

 

0.32

0.00828

4

0.0331

10

 

1.0

0.0325

4

0.130

13

*                             =             duplicate sample stored frozen prior to analysis

LOQ                      =             Limit of Quantification

-                              =             not applicable

For reporting purposes, x has been calculated from c retrospectively.

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item has been investigated and gave the following results based on the time-weighted mean measured test concentrations:

Time Point (Hours) EC50 (mg/L) 95% Confidence Limits (mg/L) NOEC (mg/L) LOEC (mg/L)
48 0.20 0.12 - 0.32 0.0073 0.017
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:

Information provided by the Sponsor indicated the test item has limited solubility in water. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

 

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.33 mg/L was obtained from a solvent spike solution method of preparation indicating this to be the limit of water solubility of this item in deionized reverse osmosis water.

 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L for 48 hours at a temperature of approximately 20°C under semi-static test conditions. The test item solution was prepared by dispersing 500µLof a 100 mg/10 mL solvent stock solution in 5 liters of test water with the aid of magnetic stirring for approximately 10 minutes. After stirring any undissolved test item was removed by filtration (0.2 µm Gelman Acrocap filter, first approximate 500 mL discarded in order to pre-condition the filter) to give the 1.0 mg/L nominal concentration. Dilutions were then prepared from this test concentration to give the remaining concentrations of 0.010, 0.032, 0.10 and 0.32 mg/L. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

Results:

Analysis of the freshly prepared 0.032, 0.10, 0.32 and 1.0 mg/L test preparations at 0 and 24 hours showed measured test concentrations to range from 0.00620 to 0.351 mg/L. A decline in measured test concentration was observed in the old or expired test media at 24 and 48 hours to between 0.00523 and 0.164 mg/L and hence it was considered appropriate to calculate the results based on the time-weighted mean measured test concentration only in order to give a “worst case” analysis of the data.

.

Exposure of Daphnia magna to the test item gave the following results based on the time-weighted mean measured test concentrations:

 

Time Point

(Hours)

EC50
(mg/L)

95% Confidence Limits (mg/L)

No Observed Effect Concentration (NOEC) (mg/L)

Lowest Observed Effect Concentration (LOEC) (mg/L)

48

0.20

0.12

-

0.32

0.0073

0.017