Methyl hydrogen octadecylphosphonate

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:

November 28, 2005 - January 12, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Methyl hydrogen octadecylphosphonate

EC Number:

246-905-6

EC Name:

Methyl hydrogen octadecylphosphonate

Cas Number:

25371-55-5

Molecular formula:

C19H41O3P

IUPAC Name:

methoxy(octadecyl)phosphinic acid

impurity 1

Reference substance name:

Octadecylphosphonic acid

EC Number:

225-216-4

EC Name:

Octadecylphosphonic acid

Cas Number:

4724-47-4

Molecular formula:

C18H39O3P

IUPAC Name:

octadecylphosphonic acid

impurity 2

Reference substance name:

Dimethyl octadecylphosphonate

EC Number:

246-904-0

EC Name:

Dimethyl octadecylphosphonate

Cas Number:

25371-54-4

Molecular formula:

C20H43O3P

IUPAC Name:

dimethyl octadecylphosphonate

Test material form:

solid

Details on test material:

Test Item Identification: X#15249Batch: 04055A001Purity: >91%Description: White waxy solid blockStorage conditions: room temperature in the dark

Specific details on test material used for the study:

Test Item Identification: X#15249Batch: 04055A001Description: White waxy solid blockStorage conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the 50 mg/25 ml solvent stock solution, solvent control (replicates R1 - R3 pooled) and the 0.20 mg/1 test group (replicates R1 - R3 and R4 - R6 pooled) at 0 hours for quantitative analysis. Duplicate samples were taken at 0 hours and stored at approximately -20°C for further analysis if necessary. Given that no test material was detected in the 0.20 mg/1 test samples at 0 hours it was considered unnecessary to provide samples for analysis at 72 hours.

Test solutions

Vehicle:

yes

Remarks:

dimethylformamide

Details on test solutions:

No definitive experimental determination of the water solubility was possible due to the surface active properties of the test material. However, testing indicated that the water solubility of the major component of the test material was less than or equal to 2.2E-04 g/1. Preliminary solubility work showed that the highest attainable test concentration (by visual inspection) that could be prepared was 0.20 mg/1 by spiking reconstituted water with an aliquot (100 ul/l) of a solvent stock solution prepared in dimethylformamide. At higher test concentrations precipitation of the test material was observed on addition of the test material solvent stock solution to water.Based on this information the test material fell into the category of 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 and whether prolonged stirring of the preparations increased the dissolved test material concentration. An amount of test material (100 mg) was dissolved in dimethyformamide and the volume adjusted to 50 ml to give a 100 mg/50 ml solvent stock solution. An aliquot (500 ul) of this solvent stock solution was dispersed in 5 litres of culture medium with the aid of magnetic stirring for approximately 10 minutes to give the required test concentration of 0.20 mg/L.The range finding test used nominal concentrations of 0.020 and 0.02 mg/L test substance with control and solvent control groups. The solvent control group was exposed to 100 uL/L of dimethylformamide. The test material was suspected to adhere to glassware. Therefore, in order to saturate any active sites, the test vessels were pre-conditioned with the test concentration to be used in the test, approximately 24 hours prior to the test start.Based on the results of the range finding test, the definative test was a "limit" test at 0.20 mg/L (nominal). An amount of test material (100 mg) was dissolved in dimethyformamide with the aid of ultrasonication for approximately 2 minutes and the volume adjusted to 50 ml to give a 100 mg / 50 ml solvent stock solution. An aliquot (200 ul) of the 100 mg / 50 ml solvent stock solution was dispersed in a final volume of 2 litres of reconstituted water to give the 0.20 mg/L test concentration.The control and solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 ul/L of dimethyformamide.

Test organisms

Details on test organisms:

TEST ORGANISM- Strain: CCAP 276/20.- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.- Method of cultivation: Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 21 ± 1 °C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ± 1 °C

pH:

7.4 to 8.5

Nominal and measured concentrations:

Nominal concentrations of 0.02 mg/L and 0.2 mg/L for the range-finding test and 0.2 mg/L for the definitive test

Details on test conditions:

In the range-finding test and the definitive test, 250 mL glass conical flasks were used. In the definitive test, six flasks each containing 100 mL of test preparation were used for the treatment group and three flasks each containing 100 mL were used for the control and solvent control groups.The control and the solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 μl of dimethylformamide.Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 4.84E+06 cells per mL. This suspension was diluted to a cell density of 9.92E+03 cells per mL prior to use. At initiation of the test the culture contained a nominal cell density of 1E+04 cells per mL.The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 150 rpm for 72 hours.Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.For the range-finding test all test vessels were pre-conditioned with the appropriate test concentration 24 hours prior to the start of the test. This was because the test material was suspected to adhere to glassware. However, no evidence of adsorption to glass was found in the preliminary recovery analyses conducted, therefore it was considered unnecessary to precondition the glassware for the definitive test.GROWTH MEDIUM- Standard medium used: Yes.OTHER TEST CONDITIONS- Adjustment of pH: No.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.- Physico-chemical measurements: The pH of the control, solvent control and test flasks was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.TEST CONCENTRATIONS- Range finding study: Yes.- Test concentrations: 0.02 and 0.2 mg/L nominal concentrations.- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Statistical analysis of the area under the growth curve data was carried out for the solvent control and 0.20 mg/L test group using a Student's t-test incorporating Bartlett's test for homogeneity of variance. There were no statistically significant differences (P > 0.05) between the solvent control and 0.20 mg/1 test group and therefore the "No Observed Effect Concentration" (NOEC) was 0.20 mg/L. The test concentration of 0.20 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guideline.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Accordingly the following results were determined from the data:ErC50 (0 - 72 h) : > 0.20 mg/1EbC50 (72 h): > 0.20 mg/1where EbC50 is the test concentration that reduced biomass by 50% and ErC50 is the testconcentration that reduced specific growth rate by 50%.No Observed Effect Concentration (NOEC) = 0.20 mg/L

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201, "Alga, Growth Inhibition Test".

 

Following a preliminary range-finding test,Scenedesmus subspicatuswas exposed to an aqueous solution of the test material at a nominal concentration of 0.20 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. No inhibition of algal growth was observed in either the range-finding or the definitive test.

 

Exposure of Scenedesmus subspicatusto the test material gave EC50 values of greater than 0.20 mg/L (nominal) and correspondingly the No Observed Effect Concentration was 0.20 mg/L (nominal).

 

The test concentration of 0.20 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent, and having due regard to the amount of auxiliary solvent permitted in the test under the OECD Guidelines. Preliminary work conducted using the analytical method supplied by the Sponsor for this test material gave a limit of quantitation (LOQ) of 20 mg/1 which meant that the test concentration of 0.20 mg/1 would not be detected. As no measured concentrations could be obtained for the test samples the results are based on nominal test concentrations only.