alpha-[butyl(dimethylsilyl)]-omega-[3-(2-{[2- (methacryloyloxy)ethyl]carbamoyloxy}ethoxy) propyl]poly[oxy(dimethyl)silylene]

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 study was conducted between 19 July 2011 and 13 August 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

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. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test item in cases where the test item is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess of test item in culture medium for a period of 24 hours prior to removing any undissolved test item present by filtration (0.2 µm Sartorius Sartopore, first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution of the test item

GLP compliance:

yes

Test material

Test material information

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control (replicates R1 – R4 pooled) and the 100% v/v saturated solution test group (replicates R1 – R2 and R3 – R4 pooled) at 0 and 48 hours for quantitative analysis. All 0-Hour samples were stored at approximately -20 degree C prior to analysis.

Duplicate samples were taken and stored at approximately -20 degree C for further analysis if necessary.

The method of analysis, recovery and test preparation analyses are described in Appendix 6 in attached section.

Test solutions

Vehicle:

no

Details on test solutions:

Pre-study media preparation trial
Information provided by the Sponsor indicated the water solubility of the test item to be less than 6.89 x 10-8 g/l. Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item 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 item under test conditions (see Appendix 5 in attached section).

Range-finding test
The results obtained from the pre-study media preparation trial conducted indicated that a saturated solution method of preparation followed by the removal of any undissolved test item by filtration was most appropriate for this test item.
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 1.0, 10 and 100% v/v saturated solution.
An amount of test item (550 mg) was dispersed in 11 litres of reconstituted water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give the 100% v/v saturated solution test concentration. A series of dilutions was made from this saturated solution to give further test concentrations of 10 and 1.0% v/v saturated solution.
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 degree C to 22 degree 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 in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification. 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 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°C prior to analysis.

Definitive test
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration no immobilisation or adverse reactions to exposure were observed.

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 were maintained in 150 ml glass beakers containing Elendt M7 medium (see Appendix 2 in attached section) in a temperature controlled room at approximately 21 degree 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 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 Water

Reconstituted Water
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

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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

Temperature was maintained at 20 degree C to 22 degree C 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 the temperatures were within the test guideline specification.

The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

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 reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.

Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration was measured using a
dissolved oxygen meter.

See physico-chemical measurements results table in any other information on methods.

Salinity:

freshwater used.

Nominal and measured concentrations:

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 1.0, 10 and 100% v/v saturated solution.

Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the
highest attainable test concentration no immobilisation or adverse reactions to exposure were observed.

Details on test conditions:

Experimental preparation
An amount of test item (500 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give the required test concentration of 100% v/v saturated solution.
The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

The concentration of the test item in the test preparation was verified by chemical analysis at 0 and 48 hours (see Appendix 6 in attached section).

Due to a technical oversight the quantity of test item weighed out for use in the definitive test (500 mg) differed to that weighed out for use in the range-finding test (550 mg).

This was considered to have had no adverse effect on the outcome of the definitive test as due to the test item’s extremely low water solubility, saturation of the test media would have occurred at either of the concentrations employed.

Exposure conditions

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 and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 20 degree C to 22 degree 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.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: Not stated

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other:

Remarks on result:

other: 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 - in section any other information on results.

No immobilisation was observed at the test concentrations of 1.0, 10 and 100% v/v saturated solution.

Based on this information, a single test concentration of four replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the highest attainable test concentration no immobilisation or adverse reactions to exposure were observed.

Chemical analysis of the test preparations at 0 and 48 hours (see Appendix 6) showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.22 mg/l. This does not infer that no test item was in solution just that which was so was at a concentration of less than 0.22 mg/l.

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 in section any
other information on results.
There was no immobilisation in 20 daphnids exposed to a test concentration of 100% v/v saturated solution for a period of 48 hours. Inspection of the immobilisation data gave the following results:

Time (h) EC50 (% v/v Saturated Solution)
24 >100
48 >100

The No Observed Effect Concentration after 24 and 48 hours exposure was 100% v/v saturated solution. The No Observed Effect Concentration is
based upon zero immobilisation at this concentration.

Observations on test item solubility
All control and test preparations were observed to be clear colourless solutions throughout the duration of the test.

Results with reference substance (positive control):

A positive control (Harlan Laboratories Ltd., Project No: 41102080) 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:

Time (h)
EC50 (mg/l) 95% Confidence limits
(mg/l)
24 1.5 1.3 - 1.8
48 0.99 0.85 - 1.1

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.

Reported statistics and error estimates:

Not Applicable

Any other information on results incl. tables

Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1:

Table1              Cumulative Immobilisation Data in the Range-finding Test

Nominal Concentration (% v/v Saturated Solution)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
1.0	0	0
10	0	0
100	0	0

 

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

Table 2              Cumulative Immobilisation Data in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)		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	1	5
	R2	0			1*
	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

*Single immobilised daphnid considered to be due to natural causes as no further immobilisation was observed.

Applicant's summary and conclusion

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% v/v saturated solution.

The No Observed Effect Concentration was 100% v/v saturated solution.

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 that the test item was insoluble in water

(water solubility less than 6.89 x 10^-8g/l). Pre-study solubility 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 pre-study media preparation trial indicated that the use of a saturated solution method of preparation followed by the removal of any undissolved test item by filtration was the most appropriate method of preparation for this test item.

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% v/v saturated solution for 48 hours at a temperature of 20°C to 22°C under static test conditions. The test item solution was prepared by stirring an excess (45 mg/l) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results. 

The 48-Hour EC₅₀for the test item to Daphnia magna based on nominal test concentrations was greater than 100% v/v saturated solution. The No Observed Effect Concentration was 100% v/v saturated solution.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.22 mg/l. This does not infer that no test item was in solution just that which was present was so at a concentration below 0.22 mg/l.

This study showed that there were no toxic effects at saturation.