[No public or meaningful name is available]

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:

2022

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

not specified

Principles of method if other than guideline:

This study was performed in order to evaluate the toxicity of tributyl(ethyl)phosphonium
diphenyl phosphate towards Desmodesmus subspicatus. This freshwater green alga was
chosen as a typical species of phytoplankton.
The system response is the reduction of growth in a series of algal cultures (test units) exposed to the test item. This response was evaluated as a function of the exposure concentration in comparison with the average growth of the replicates, unexposed blank control
cultures. For full expression of the system response to toxic effects (optimal sensitivity), the
cultures were allowed unrestricted exponential growth under nutrient sufficient conditions
and continuous light for a sufficient period of time to measure reduction of the specific growth
rate.
The resulting endpoints are the NOEC (no observed effect concentration) and the LOEC
(lowest observed effect concentration) and, if applicable, the EC10 and EC50 for growth rate
and yield.
A positive control was tested in a separate study to assure that the test conditions are reliable.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tributyl(ethyl)phosphonium diphenyl phosphate

Cas Number:

2409816-82-4

Molecular formula:

C26H42O4P2

IUPAC Name:

Tributyl(ethyl)phosphonium diphenyl phosphate

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

not specified

Remarks:

An analytical method for the determination of tributyl(ethyl)phosphonium diphenyl phosphate using IC was validated. The method is fully described in validation report VB21062908G926. This document and the corresponding raw data will be archived followi

Details on sampling:

Sample Preparation
The 46 and 100 mg/L test item solutions were diluted as follows:
100 mg/L: 50 µL of 100 mg/L solution were mixed with 950 µL demineralised water resulting in a 20fold dilution;
46 mg/L: 250 µL of 46 mg/L solution were mixed with 750 µL demineralised water resulting in a 4fold dilution

Test solutions

Vehicle:

not specified

Details on test solutions:

The concentration of the test item in the test solutions will be determined as follows:
Linear calibration curve:
cmeasured [mg/L] =[ (Measured value - a)/
b] *[ 100 %/QC Recovery Rate [%]] ∗ [dilution factor]
with
a = intercept
b = slope

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Unicellular freshwater green alga.
Genus Desmodesmus
Species subspicatus
SAG Strain Number 86.81
Taxonomic position Chlorophyta - Chlorophyceae

Study design

Total exposure duration:

72 h

Test conditions

Test temperature:

19.4 – 20.3 °C

Details on test conditions:

Duration: 72 hours
Treatments: 100 / 46 / 22 / 10 / 4.6 mg/L (nominal)
The concentrations to be tested are based on a non GLP
pre-test.
Temperature: 19.4 – 20.3 °C
Lighting: within the specified range (4440 – 8880 Lux)
Measurement: cell number via electronic cell counter (Measurement
range: 5 – 12 µm)
Replicates
Treatments: 3 vessels, each filled with 45 ±1 mL test solution and algae
1 vessel, filled with 45 ±1 mL test solution without algae
for analytical determination
Blank Control: 6 vessels, each filled with 45 ±1 mL algal medium and algae
1 vessel filled with 45 ±1 mL algal medium without algae
for analytical determination

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

26.31 mg/L

95% CI:

>= 7.6 - <= 95.61

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.02 mg/L

95% CI:

>= 1.29 - <= 12.52

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

19 mg/L

95% CI:

>= 11.51 - <= 31.36

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 7

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Calculation of results was performed with the help of validated software (Microsoft Excel®).
The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.3.0. The details of calculation are stated in Annex 5: Statistical calculation
using ToxRat® Professional 3.3.0.
The following values were determined.
Table 10 Biological Results of the Test Item
Parameter Value 95 % confidence interval
NOEC (Growth Rate)72 h 4.60 mg/L --
NOEC (Yield) 72 h 4.60 mg/L --
LOEC (Growth Rate) 72 h 10.00 mg/L --
LOEC (Yield) 72 h 10.00 mg/L --
72h ErC10 19.00 mg/L 11.51 – 31.36 mg/L
72h EyC10 4.02 mg/L 1.29 – 12.52 mg/L
72h ErC50 > 100 mg/L* --
72h EyC50 26.31 mg/L 7.06 – 95.61 mg/L
* For the endpoint growth rate, an EC50 of 213.05 mg/L was extrapolated by the program.
This value lies fare above the highest tested concentration 100 mg/L Therefore for the
endpoint growth rate the EC50 was reported as the range > 100 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

One valid experiment was performed.

Conclusions:

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth
rate 0.64 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

Executive summary:

One valid experiment was performed.
The study was performed using 5 concentrations ranging from 4.6 to 100 mg/L (nominal).
Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 ± 1 hours
with an electronic cell counter. Growth rate µ and the yield1 were determined from the cell
number at the respective observation times.
Significant inhibition of algal growth was observed at all tested concentrations with the exception of the lowest concentrated treatment 4.6 mg/L.
At the start and at the end of the test, the content of the test item in the test solutions was
determined using IC.
The measured concentrations lay between 98 % and 102 % of the nominal concentrations
at the beginning of the test and between 100 % and 107 % of the nominal concentrations at
the end of the test. Therefore, the determination of the biological results was based on the
nominal concentration (see Guidance Doc. No.23 (§176)).
The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth
rate 0.64 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).
The following results for the test item tributyl(ethyl)phosphonium diphenyl phosphate were
determined:

Table 3 Results of the test item
Endpoint                    NOEC                 LOEC                EC10                     EC50
Growth Rate               4.60 mg/L          10.00 mg/L      19.00 mg/L             > 100.00 mg/L
Yield                          4.60 mg/L          10.00 mg/L       4.02 mg/L              26.31 mg/L
Note: The NOEC is determined by comparing each treatment with the blank control using a
hypothesis test. A statistically insignificant variation is considered as "no observed effect".
In contrast, EC10 and EC50 are determined by regression analysis and read from the toxicity
vs. concentration graph.