Potassium hexadecyl hydrogen phosphate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-11-19 to 2012-11-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is regarded as reliable without restrictions because it was conducted in compliance with GLP regulation and guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 2006-03-23

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

cited as: Commission Regulation (EC) No 761/2009, Annex, Part C, C.3 dated 2009-08-24

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L
- Sampling method: Duplicate samples from the freshly prepared test medium (without algae) of the only test concentration and from the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test medium of the only test concentration and the control (without algae) were taken at the end of the test (after the 72 hours test period).
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ -10 °C), protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Stock Solution: The test item was used to prepare the stock solution. 50.39 mg test item were dissolved in 50 mL methanol by ultrasonification for 10 min and stirring for 1 min to obtain a stock solution of approximately 1 g test item/L.
Standard Solutions: Appropriate amounts of the stock solution were diluted with a mixture of acetonitrile / diluted-test water (1/1, v/v) to obtain standard solutions in the range from 0.005 to 0.15 mg test item/L.

Dosage of Test Item:
The test item was not well soluble in test water. Therefore, a stock suspension of 100 mg test item/L was prepared by suspending 199.1 mg test item in 1991 mL test water. The stock suspension was stirred for 24 hours at room temperature in the dark to dissolve as much test item as possible. Then, non-dissolved fractions of the test item were separated by extracting the test water from below the water surface followed by membrane filtration (0.45 µm cellulose nitrate filter). The test media were prepared just before introduction of the algae (= start of the test).

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: No. 61.81 SAG
- Source: "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", 37073 Göttingen, Germany.
- Method of cultivation: The algae were cultivated in the laboratories of IBACON under standardised conditions according to the test guidelines.

ACCLIMATION
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

22 - 23 °C

pH:

8.0 to 9.0 - 9.1

Nominal and measured concentrations:

A filtrate of a supersaturated stock suspension of nominal 100 mg/L. No measured concentrations are available (below LOD).

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 50 mL volume with 50 mL of test medium.
- Initial cells density: biomass of 5000 algal cells per mL test medium
- Control end cells density: 114.827
- No. of vessels per concentration: 6
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- in accordance to the guideline

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuously illumination
- Light intensity and quality: 5883 lux (range: 5680 to 6110 lux)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations:

The cell densities in the samples were determined by spectrophotometrical measurement. Therefore defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced.
The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae).
The cell densities in a number of samples from one control replicate were counted by microscope after 72 hours of test duration. Out of this control five defined dilutions were prepared and measured spectrophotometrically. Based on the counted cell densities and the absorption of the control sample and the dilutions a linear regression was performed for the calculation of the cell densities in all other samples measured spectrophotometrically during the test.

TEST CONCENTRATIONS
- Range finding study:
For the determination of the test concentrations a range-finding test was performed. However, concentrations in excess of the solubility were not tested.
- Test concentrations: 100 mg/L

Reference substance (positive control):

yes

Remarks:

dipotassium chromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing at a nominal test concentration of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the only concentration tested.

Results with reference substance (positive control):

- Results with reference substance valid: yes
- EC50: 0.471 (SD 0.423 - 0.527) mg/L Biomass; 0.904 (SD 0.867 - 0.941) mg/L growth rate
- 72-hour NOEC: 0.21 mg/L
- Reference Item Potassium Dichromate in August 2012

Validity criteria fulfilled:

yes

Conclusions:

The 72-h EC50 value based on growth rate was determined to be greater than 100 mg/L and based on yield was determined to be greater than 100 mg/L. The 72-h EC10 value based on growth rate was determined to be greater than 100 mg/L and based on yield was determined to be greater than 100 mg/L. The overall NOEC was determined to be greater than 100 mg/L. The results are based on the nominal concentrations.

Executive summary:

The purpose of this study was to determine the inhibitory effect of the test item potassium hexadecyl hydrogen phosphate on the growth of the freshwater green algae Pseudokirchneriella subcapitata according to OECD TG 201 and Regulation (EC) No 440/2008 method C.3. The test was performed as static limit test. Exponentially growing cultures of this unicellular green algal species were exposed to one concentration of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.

Test concentrations were prepared by appropriate diluting of a stock solution. Based upon the results from the preliminary experiment, nominal concentrations of 100 mg/L were investigated in the main study. As the solubility of the test item in aquatic test media is very low, the concentrations of dissolved test item were below the Limit of Detection (0.7 µg test item/L). However, all reported results refer to nominal loading rate.

The 72-hour NOEC was determined to be at least the filtrate of nominal 100 mg test item/L (nominal). The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L. The 72-hour LOEC and the 72-hour EC50 were clearly higher than the filtrate of nominal 100 mg test item/L (nominal). These values could not be quantified due to the absence of toxicity of the test item up to the filtrate of nominal 100 mg test item/L (nominal).

The 72-h EC50 value based on growth rate was determined to be greater than 100 mg/L and based on yield was determined to be greater than 100 mg/L. The 72-h EC10 value based on growth rate was determined to be greater than 100 mg/L and based on yield was determined to be greater than 100 mg/L. The overall NOEC was determined to be greater than 100 mg/L. The results are based on the nominal concentrations.

Description of key information

The 72-h EC50 value based on growth rate was determined to be greater than 100 mg/L and based on yield was determined to be greater than 100 mg/L. The 72-h EC10 value based on growth rate was determined to be greater than 100 mg/L and based on yield was determined to be greater than 100 mg/L. The overall NOEC was determined to be greater than 100 mg/L. The results are based on the nominal concentrations.

Additional information

The purpose of this test was to determine the inhibitory effect of the test item potassium hexadecyl hydrogen phosphate on the growth of the freshwater green algae Pseudokirchneriella subcapitata according to OECD TG 201 and Regulation (EC) No 440/2008 method C.3. The test was performed as static limit test. Exponentially growing cultures of this unicellular green algal species were exposed to one concentration of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.

Test concentrations were prepared by appropriate diluting of a stock solution. Based upon the results from the preliminary experiment, nominal concentrations of 100 mg/L were investigated in the main study. As the solubility of the test item in aquatic test media is very low, the concentrations of dissolved test item were below the Limit of Detection (0.7 µg test item/L). However, all reported results refer to nominal loading rate.

The 72-hour NOEC was determined to be at least the filtrate of nominal 100 mg test item/L (nominal). The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L. The 72-hour LOEC and the 72-hour EC50 were clearly higher than the filtrate of nominal 100 mg test item/L (nominal). These values could not be quantified due to the absence of toxicity of the test item up to the filtrate of nominal 100 mg test item/L (nominal).

The 72-h EC50 value based on growth rate was determined to be greater than 100 mg/L and based on yield was determined to be greater than 100 mg/L. The 72-h EC10 value based on growth rate was determined to be greater than 100 mg/L and based on yield was determined to be greater than 100 mg/L. The overall NOEC was determined to be greater than 100 mg/L. The results are based on the nominal concentrations.