Phosphorous acid, mixed 2,4-bis(1,1-dimethylpropyl)phenyl and 4-(1,1-dimethylpropyl)phenyl triesters

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 December 2009 to 22 January 2010.

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.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 15/09/09 Date of signature: 26/11/09

Test material

Test material information

Constituent 1

Reference substance name:

[4-(2-methylbutan-2-yl)phenyl]x [2,4-bis(2-methylbutan-2-yl)]3-x phosphite (where x = 0, 1, 2, 3)

IUPAC Name:

[4-(2-methylbutan-2-yl)phenyl]x [2,4-bis(2-methylbutan-2-yl)]3-x phosphite (where x = 0, 1, 2, 3)

Details on test material:

Sponsor's identification :DVS005u (aka Weston 705)
Description : clear colourless viscous liquid
Batch number : MW9F23A901 (range-finding and definitive test preparations
T167-20090615 (preliminary recovery and stability analyses and standard solution preparations)
Date received : 5 November 2009
Expiry date : 15 June 2010
Storage conditions :approximately 4°C under nitrogen in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/l loading rate WAF test group (replicates R1 - R3 and R4 - R6 pooled) and control at 0 and 72 hours

- Sampling method: A volume of test sample was extracted with dichloromethane (3 x 50 ml). The extracts were filtered through anhydrous sodium sulphate. The combined extracts were evaporated to dryness and the residue re-dissolved in acetonitrile.




- Sample storage conditions before analysis: Duplicate samples were taken at 0 hours and stored at approximately 20ºC for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Validation of mixing period
Pre-study WAF validation work conducted for the Acute Toxicity to Daphnia magna study (section 6.1.3 of IUCLID dossier) indicated that the maximum amount of dissolved test material present in a 100 mg/l loading rate WAF was achieved after a preparation period of 120 hours. However, discussion with the Sponsor indicated that the test material hydrolysed with prolonged stirring and therefore a preparation period of 24 hours was considered appropriate.

- Eluate: same as culture media

- Differential loading: Not in the definitive test

- Controls: The control group was 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(s) including control(s)): not applicable

- Evidence of undissolved material (e.g. precipitate, surface film, etc): Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland, UK.
- Age of inoculum (at test initiation): not stated in report
- Method of cultivation: Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 10ˆ3 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 10ˆ4 – 10ˆ5 cells/ml.

ACCLIMATION
- Acclimation period: not stated in report
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: not applicable

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

All test and control cultures were inspected microscopically at 72 hours.

Test conditions

Hardness:

Not stated in report

Test temperature:

24 ± 1°C.
The temperature within the incubator was recorded daily.

pH:

The pH values of the control cultures were observed to increase from pH 7.3 at 0 hours to pH 7.6 – 7.7 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Dissolved oxygen:

Not stated in report

Salinity:

Not stated in report

Nominal and measured concentrations:

Range finding test: 10 and 100 mg/l loading rate
Definitive study: 100 mg/l loading rate

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: closed - plugged with polyurethane foam bungs to reduce evaporation.
- Material, size, headspace, fill volume of each vessel: 250 ml glass conical flasks each containing 100 ml of test preparation
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable, as static test conditions
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 4 x 10^3 cells / ml
- Control end cells density: 7.03 x 10^5 cells/ml
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): Six flasks
- No. of vessels per control (replicates): Six flasks
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis purified deionised water (Elga Optima 15+).
-NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

- Culture medium different from test medium: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
- Intervals of water quality measurement: not stated in report

OTHER TEST CONDITIONS
- Sterile test conditions: not stated in report
- Adjustment of pH: pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCl.
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 7000 lux
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
- Chlorophyll measurement: No
- Other: none

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable, as only one concentration used in definitive test
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: 10 and 100 mg/l loading rate
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at 10 and 100 mg/l loading rate WAF.
Based on this information a single loading rate of six replicates of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): There were no abnormalities detected in any of the control or test cultures.
- Unusual cell shape: No
- Colour differences: At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be pale green dispersions.
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At both the start and end of the mixing period and following a 1-Hour standing period the WAF was observed to have formed a clear colourless media column with an oily layer of test material floating at the media surface. Microscopic examination of the WAF showed there to be no globules or micro-dispersions of test material to be present.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes

- EC50: ErC50 (0 – 72 h) : 0.49 mg/l*
EyC50 (0 – 72 h) : 0.18 mg/l, 95% confidence limits 0.16 – 0.21 mg/l
No Observed Effect Concentration (NOEC) based on growth rate: 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on yield rate: 0.0625 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield rate: 0.125 mg/l
The results from the positive control with potassium dichromate were within the normal ranges for this reference material.

Reported statistics and error estimates:

A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/l loading rate to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Any other information on results incl. tables

 Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 43 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours                      :   4.47 x 10³cells per ml
Mean cell density of control at 72 hours                   :   1.94 x 10⁵cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 18% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

Temperature was maintained at 24 ± 1ºC throughout the test.

The pH values of the control cultures were observed to increase from pH 7.3 at 0 hours to pH 7.6 – 7.7 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Desmodesmus subspicatus to the test material gave EL*50 values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.13 to 0.15 mg/l. A slight decline in measured test concentrations was observed at 72 hours in the range of 0.067 to 0.12 mg/l. This decline was in line with the preliminary stability analyses conducted which indicated that the test material was unstable in culture medium over the test period at a concentration of 0.10 mg/l.
Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, the results were based on nominal loading rates only.

Executive summary:

Introduction. A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus.  The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 440/2008.

Methods.  Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to a Water Accommodated Fraction (WAF) of the test material, at a single nominal loading rate of 100 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

Results. Exposure of Desmodesmus subspicatus to the test material gave EL*₅₀ values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

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

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.13 to 0.15 mg/l. A slight decline in measured test concentrations was observed at 72 hours in the range of 0.067 to 0.12 mg/l. This decline was in line with the preliminary stability analyses conducted which indicated that the test material was unstable in culture medium over the test period at a concentration of 0.10 mg/l.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, the results were based on nominal loading rates only.

*EL = Effective Loading Rate