Phosphoric acid, mono and bis-linear butyl esters, potassium salts

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:

2017-07-10 - 2017-07-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

other: EPA Ecological Effects Test Guidelines, OCSPP 850.5400, Algal Toxicity, Tiers I and II

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch/Lot Number: 7675229
Description: White/gray solid
Expiry date: 31 August 2018

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control and 100 mg/L solutions
- Sampling method: Duplicate samples (from 3 replicates) taken at t=0 ad t=72h in plastic tubes.
- Sample storage conditions before analysis: Frozen and kept at approx. -20 deg C at the test facility. One set of samples sent to the test site for analysis and one set retained as back-up at the test facility.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution with a nominal concentration of 100 mg/L was prepared with direct addition of the test item, mixed into the test medium (OECD Medium) using ultrasonicbath (approximately 4 minutes). As a Limit Test was carried out, further dilution of stock solution was not be performed.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY.
- Method of cultivation: Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.

ACCLIMATION
- Culturing media and conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines. The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, after an incubation period of three days.
- Any deformed or abnormal cells observed: When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

not applicable

Hardness:

no data

Test temperature:

22.8 – 23.0 °C

pH:

7.40 – 8.92 during the experiment

Dissolved oxygen:

no data

Salinity:

no data

Conductivity:

no data

Nominal and measured concentrations:

nominal: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: glass, 250 mL. Fill volume = 100 mL.
- Aeration: fitted with air-permeable stoppers

- Initial cells density: approximately 1E04 cells/mL
- Control end cells density: 69.67E04 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: The algal culture flasks were continuously illuminated.
- Light intensity and quality: The light intensity at the position occupied by algal culture flasks during the test was about 8335 lux (equivalent to ~113 μE/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm).


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
- Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable - limit test
- Justification for using less concentrations than requested by guideline: Based on results of range-finding study.
- Range finding study
- Test concentrations: 0.1, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study:
Test concentration (mg/L)/ Average number of cells at 72 hours (E04 cell/mL): 0.1/72.00; 1/70.00; 10/70.50; 100/70.00
Because no toxic effect was observed during the preliminary range-finding test, only one test concentration of the test item in the test medium (100 mg/L) and one control group were tested in a Limit Test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

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:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

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 any observed morphological deviations during the experiment.
- Unusual cell shape: no
- Colour differences: no
- 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: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate
satisfactory test conditions.
The date of the last study (Study Code: 17/058-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 07 – 10 March 2017.
The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EyC 50: 0.54 mg/L, (95 % confidence limits: 0.50 – 0.59 mg/L)
The 72h EbC 50: 0.64 mg/L, (95 % confidence limits: 0.59 – 0.70 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Validity criteria fulfilled:

yes

Conclusions:

72h EbC50 value (biomass): > 100 mg/L (nominal)
72h ErC50 value (growth rate): > 100 mg/L (nominal)
72h EyC50 value (yield): > 100 mg/L (nominal)
72h NOEC: 100 mg/L (nominal)
72h LOEC: > 100 mg/L (nominal)

Executive summary:

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an

exposure period of 72 hours (OECD TG 201).

Because a toxic response was not observed during the preliminary concentration rangefinding test, a Limit Test was carried out using only one test concentration of 100 mg/L (nominal) and one control group.

The test concentration was analytically determined at the start and at the end of the experiment. Measured concentration was 105.6 mg/L at the start and 106.3 mg/L at the end of the experiment. As the analytically measured concentration deviated not more than 20 per cent from the nominal, the biological results are based on the nominal test item concentration.

The test design included six replicates at test concentration and six replicates for the untreated control.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (α = 0.05) by TOXSTAT software.

The ErC50, EbC50 and EyC50 values of the test item were determined directly from the raw data.

Under the conditions of this algal growth inhibition test the calculated endpoints for the effect of the test item salt were the following:

72h ErC50 value (biomass): > 100 mg/L (nominal)

72h EbC50 value (growth rate): > 100 mg/L (nominal)

72h EyC50 value (yield): > 100 mg/L (nominal)

72h NOEC: 100 mg/L (nominal)

72h LOEC: > 100 mg/L (nominal)

Description of key information

72h ErC50 value (biomass): > 100 mg/L (nominal)

72h EbC50 value (growth rate): > 100 mg/L (nominal)

72h EyC50 value (yield): > 100 mg/L (nominal)

72h NOEC: 100 mg/L (nominal)

72h LOEC: > 100 mg/L (nominal

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours (OECD TG 201).

Because a toxic response was not observed during the preliminary concentration rangefinding test, a Limit Test was carried out using only one test concentration of 100 mg/L (nominal) and one control group.

The test concentration was analytically determined at the start and at the end of the experiment. Measured concentration was 105.6 mg/L at the start and 106.3 mg/L at the end of the experiment. As the analytically measured concentration deviated not more than 20 per cent from the nominal, the biological results are based on the nominal test item concentration.

The test design included six replicates at test concentration and six replicates for the untreated control.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (α = 0.05) by TOXSTAT software.

The ErC50, EbC50 and EyC50 values of the test item were determined directly from the raw data.

Under the conditions of this algal growth inhibition test the calculated endpoints for the effect of the test item were the following:

72h ErC50 value (biomass): > 100 mg/L (nominal)

72h EbC50 value (growth rate): > 100 mg/L (nominal)

72h EyC50 value (yield): > 100 mg/L (nominal)

72h NOEC: 100 mg/L (nominal)

72h LOEC: > 100 mg/L (nominal)