Reaction mass of Bis(2,4-bis(2-methylbutan-2-yl)phenyl) 4-(2-methylbutan-2-yl)phenyl phosphite and 2,4-Bis(2-methylbutan-2-yl)phenyl bis(4-(2-methylbutan-2-yl)phenyl) phosphite and Tris(4-(2-methylbutan-2-yl)phenyl) phosphite.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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 relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP inspection: 15/09/09 Date of Signature on GLP certificate: 26/11/09

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Sponsor's identification :DVS005u
Description : Clear colourless viscous liquid
Chemical name :Tris (amylated phenyl) phosphate mixture
Batch number : T167-20090615
Date received : 25 June 2009
Expiry date : not supplied
Storage conditions : Approximately 4°C in the dark under nitrogen

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Range finding test: Nominal loading rates of 1.0, 10 and 100 mg/l
Definitive test: Loading rates of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l

- Sampling method:
Water samples were taken from the control and the 1.0, 3.2, 10, 32 and 100 mg/l loading rate WAF test groups (replicates R1 – R2 pooled) at 0 and 48 hours for quantitative analysis.
Duplicate samples and samples of the 1.8, 5.6, 18 and 56 mg/l loading rate WAF test groups (replicates R1 – R2 pooled) were taken.

The test material concentration in the test samples was determined by high performance liquid chromatography (HPLC) using an external standard. The test material gave a chromatographic profile consisting of a number of peaks. The results have been calculated using the total peak area associated with the test material.


- Sample storage conditions before analysis: 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:
Due to the low aqueous solubility and complex nature of the test material for the purposes of the definitive test the test material was prepared as a Water Accommodated Fraction (WAF).
Amounts of test material (10, 18 ,32 ,56, 100, 180, 320, 560 and 1000 mg) were each separately added to the surface of 10 litres of reconstituted water to give the 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l loading rates respectively. The test material required for loading rates 1.0 and 1.8 mg/l was weighed onto microscope slides due to the small amount of test material required. The slides with test material on were then submerged in the water columns attached to glass rods. The test material required for loading rates 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l was added directly to the water disposable plastic syringes. After the addition of the test material, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present.

The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 48 hours.
Total Organic Carbon (TOC) analysis was also performed on the test preparations at 0 and 48 hours

- Eluate:
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.


- Differential loading:
No data

- Controls:
A positive control (Harlan Laboratories Ltd Project No: 0039/1094) conducted approximately every six months used potassium dichromate as the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
An amount of reference material (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (50 ml) of this stock solution was diluted in reconstituted water and the volume adjusted to 500 ml to give a 10 mg/l stock solution. Aliquots (16, 28, 50, 90 and 160 ml) of the 10 mg/l stock solution were each separately dispersed in a final volume of 500 ml of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Exposure conditions for the positive control were similar to those used in the definitive test.
The temperature was maintained at 21°C to 22°C. Some of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the protocol. This was considered not to have affected the outcome or validity of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperature were within test guideline specification.

Control group maintained under identical conditions but not exposed to the test material.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):not applicable

- Concentration of vehicle in test medium (stock solution and final test solution): not applicable

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
None

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain: not applicable
- Source: In-house labortaory cultures
- Age at study initiation (mean and range, SD): less than 24 hours old
- Weight at study initiation (mean and range, SD): not reported
- Length at study initiation (length definition, mean, range and SD): not reported
- Valve height at study initiation, for shell deposition study (mean and range, SD): not applicable
- Peripheral shell growth removed prior to test initiation: not applicable
- Method of breeding: Parthenogenesis
- Feeding during test: no feeding during exposure period
- Food type: not applicable
- Amount: not applicable
- Frequency: not applicable

ACCLIMATION: - not applicable

QUARANTINE (wild caught) - not applicable

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Test conditions

Hardness:

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

Test temperature:

20 - 22°C

pH:

7.7 - 7.8 at 0 hours
7.8 - 8 at 48 hours

Dissolved oxygen:

Dissolved oxygen concentration measured using a dissolved oxygen meter.
The oxygen concentration in some of the test vessels at 48 hours 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 supersaturating the diluent and was considered not to have had an impact on the outcome or validity of the test as no adverse effects were observed in the control daphnids.

Salinity:

Not applicable

Nominal and measured concentrations:

Range finding test: Nominal loading rates of 1.0, 10 and 100 mg/l
Definitive test: Loading rates of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass jars
- Type: closed
- Material, size, headspace, fill volume: 250 ml glass jars containing approximately 250 ml of test preparation
- Aeration: Test vessels were not aerated
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): The test preparations were not renewed during the exposure period.
- No. of organisms per vessel: 10 daphnids
- No. of vessels per concentration (replicates): 2 (duplicates)
- No. of vessels per control (replicates): 2 (duplicates)
- No. of vessels per vehicle control (replicates): not applicable, as no vehicle
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS
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
- Adjustment of pH: not conducted during study

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

- Light intensity: not stated in report

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
- Spacing factor for test concentrations: Loading rates of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l

- Justification for using less concentrations than requested by guideline: not applicable

- Range finding study: Nominal loading rates of 1.0, 10 and 100 mg/l

- Results used to determine the conditions for the definitive study:
No immobilisation was observed at the 1.0 mg/l loading rate WAF. However, immobilisation was observed at the 10 and 100 mg/l loading rate WAFs.
Based on this information loading rates of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Based on loading rate WAF

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.78 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: control

Basis for effect:

mobility

Remarks on result:

other: 0.68 - 0.88 mg/l

Details on results:

- Behavioural abnormalities: not applicable

- Observations on body length and weight: not recorded

- Other biological observations: immbolisation only

- Mortality of control: The results from the positive control with potassium dichromate were within the normal range for this reference material.

- Other adverse effects control: none recorded

- Abnormal responses: not applicable

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none

- Effect concentrations exceeding solubility of substance in test medium: Samples of the control and 1.0, 3.2, 10, 32 and 100 mg/l loading rate WAFs were taken at 0 (fresh media) and 48 hours (old media) for Total Organic Carbon (TOC). Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that all the results were around the limit of quantitation of the analytical method.

Results with reference substance (positive control):

- Results with reference substance valid? Yes. The results from the positive control with potassium dichromate were within the normal range for the reference material.

- Mortality: not applicable - immobilisation examined

- EC50/LC50: EC50 (48-hrs) - 0.77 mg/l (sd = 0.20)

- Other: NOEC (48 hrs) = 0.32 mg/l (NOEC based upon zero immobilisation at this concentration)

Reported statistics and error estimates:

The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curves and standard errors were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (Toxcalc 1999).
Probit analysis is used when two or more partial responses to exposure are shown.

Any other information on results incl. tables

Validation of Mixing Period

Pre-study investigational work conducted for the validation of mixing period indicated that quantifiable amounts of Total Organic Carbon (TOC) were detected for a 100 mg/l loading rate WAF, after a stirring period of 23 hours with a 1-Hour standing period. Higher quantifiable amounts of TOC were detected when the mixing period was extended up to 95 hours with a 1-Hour standing period.

Further validation work conducted using extended mixing periods indicated that TOC increased for a 100 mg/l loading rate WAF over a range of stirring periods of 23, 47, 71, 95 and 119 hours with a 1-Hour standing period. 

However, preliminary testing using a 119-Hour mixing period followed by a 1-Hour standing period produced variable results and discussion with the Sponsor indicated that the test material hydrolysed over time. Therefore it was considered appropriate to conduct the test using a 23-Hour stirring period followed by a 1-Hour standing period.

Range-finding Test

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in Table1. (Please see attached).

No immobilisation was observed at the 1.0 mg/l loading rate WAF. However, immobilisation was observed at the 10 and 100 mg/l loading rate WAFs.

Based on this information loading rates of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, were selected for the definitive test.

Definitive Test

Immobilisation data

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table2.

Inspection of the immobilisation data at 24 and 48 hours based on the nominal loading rates gave the following results:

Time (h)	EL*50(mg/l)	95% Confidence limits (mg/l)
24	> 100	-
48	> 100	-

The No Observed Effect Loading rates after 24 and 48 hours exposure was 56 mg/l loading rate WAF. The No Observed Effect Loading rate is based upon zero immobilisation at this loading rate.

The results of the definitive test differ slightly to the results of the range-finding test which showed 100% immobilisation at 100 mg/l after 24 hours exposure. This difference was considered to be due to a variation in response of the daphnids and/or undissolved micro-globules of test material not detected at the start of the test, as careful examination of the data revealed no other cause for this difference in response. However this was considered not to have affected the outcome or validity of the study given that the definitive test is a more robust test.

Physico-chemical measurements

The results of the physico-chemical measurements are given in Appendix 5. (Please see attached).Temperature was maintained at 20°C to 22ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.

The temperatures at 0 hours were measured to be slightly in excess of the 20 ± 1°C given in the protocol. This was considered not to have affected the outcome or validity of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test.

The oxygen concentration in some of the test vessels at 48 hours 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 supersaturating the diluent and was considered not to have had an impact on the outcome or validity of the test as no adverse effects were observed in the control daphnids.

Vortex depth measurements

The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion (see Table 4 - please see attached).

Observations on test material solubility

Observations on the test media were carried out during the mixing and testing of the WAFs.

At the start of the mixing period the 1.0 and 1.8 mg/l loading rates were observed to be clear, colourless water columns with oily test material on the microscope slide which was submerged at the water surface. The 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l loading rates were observed to be clear, colourless water columns with oily slick of test material at the water surface. After 23 hours stirring and a 1-Hour standing period the 1.0 and 1.8 mg/l loading rates were observed to remain clear, colourless water columns with oily test material on the microscope slide. The 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l loading rates were observed to remain clear, colourless water columns with oily slick of test material at the water surface. After siphoning and for the duration of the test, the 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l loading rates were observed to be clear, colourless solutions. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present.

Chemical and total organic carbon analysis of test loading rates

Chemical analysis of the control and 1.0, 3.2, 10, 32 and 100 mg/l loading rate WAFs (see Appendix 2) at 0 and 48 hours showed measured samples ranging from less than the Limit of Quantitation (LOQ) of the analytical method (which was determined to be 0.0024 mg/l) to 0.421 mg/l.

Samples of the control and 1.0, 3.2, 10, 32 and 100 mg/l loading rate WAFs (see Appendix 3) were taken at 0 (fresh media) and 48 hours (old media) for Total Organic Carbon (TOC). Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that all the results were around the limit of quantitation of the analytical method.

Therefore, given that toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, and the dissolved test material was around the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

Positive Control

Cumulative immobilisation data from the exposure of Daphnia magna to the reference material (Harlan Laboratories Ltd Project No: 0039/1094) during the positive control are given in Table 3. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 1 and 2.

Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the 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)
3	> 3.2		-
24	1.0	0.90	-	1.2
48	0.78	0.68	-	0.88

The No Observed Effect Concentrations after 24 and 48 hours were 0.56 and 0.32 mg/l respectively. 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 7.8 (SE = 1.7) and 12 (SE = 2.4) 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 0.77 mg/l (sd = 0.20).

*EL = Effective Loading rate

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48-Hour EL*50 for the test material to Daphnia magna based on nominal loading rates was greater than 100 mg/l loading rate WAF and the No Observed Effect Loading rate was 56 mg/l loading rate WAF.

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 Regulation (EC) No. 440/2008.

Methods. Due to the low aqueous solubility and complex nature of the test material it was considered appropriate for the test material to be prepared as a Water Accommodated Fraction (WAF). 

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to Water Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l for 48 hours at a temperature of 20°C to 22°C under static test conditions. The number of immobilised Daphnia 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 EL*₅₀ for the test material to Daphnia magna based on nominal loading rates was greater than 100 mg/l loading rate WAF and the No Observed Effect Loading rate was 56 mg/l loading rate WAF.

Chemical analysis of the control and 1.0, 3.2, 10, 32 and 100 mg/l loading rate WAFs at 0 and 48 hours showed measured samples ranging from less than the Limit of Quantitation (LOQ) of the analytical method (which was determined to be 0.0024 mg/l) to 0.421 mg/l.

Samples of the control and 1.0, 3.2, 10, 32 and 100 mg/l loading rate WAFs were taken at 0 (fresh media) and 48 hours (old media) for Total Organic Carbon (TOC). Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that all the results were around the limit of quantitation of the analytical method.

Therefore, given that toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, and the dissolved test material was around the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

The 48-Hour EC₅₀ for the reference material to Daphnia magna based on nominal concentrations was 0.78 mg/l with 95% confidence limits of 0.68 – 0.88 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

*EL = Effective Loading rate