1,4-Benzenediol, 2-(6-oxido-6H-dibenz[c,e][1,2]oxaphosphorin-6-yl)-

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:

May-August 2021

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

The solubility of the test item in algal medium was determined as 1.94 mg/L. Therefore, a
stock solution containing 2.0 g/L in acetone was prepared. 400 μL of this stock solution was
mixed with 400 mL algal medium (demineralised water enriched with minerals but without
algae) for preparation of the test solution.
The concentration of the solvent was 1000-fold higher than the nominal concentration in the
treatment. For the solvent control, only acetone in a concentration of 1 mL/L was used.
Note: Because of the limited solubility of the test item in acetone, the concentration of the
solvent surpasses the recommended solvent concentration of 0.1 mL/L (as stated in OECD
guidance document no. 23). Because, for a solvent concentration of 0.1 mL/L in the test
solution, a stock solution containing 20 g/L in acetone should have been prepared, but this
was not possible, because this concentration was not soluble in acetone.
However, it was proven in several experiments, though, that a concentration

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
Origin and Culture
The culture of Desmodesmus subspicatus was obtained in May 2020 by MBM Sciencebridge
GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept
as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was
prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Test type:

static

Water media type:

freshwater

Remarks:

Demineralised water

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22.4 – 23.8 °C

pH:

8.5

Nominal and measured concentrations:

nominal 2 mg/L
measured 0.12 mg/L

Details on test conditions:

Date: 25. – 28. May 2021
Treatments: 2 mg/L nominal concentration
The concentrations to be tested are based on non a GLP
pre-test.
Vessels: glass flasks total volume 65 mL
Duration: 72 ± 1 hours
Lighting: within the specified range (4440 – 8880 Lux)
Temperature: 22.4 – 23.8 °C
Replicates
Treatments: 6 vessels, each filled with 45 ±1 mL spiked test solution and
algae
1 vessel, filled with 45 ±1 mL spiked 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
Solvent Control: 6 vessels, each filled with 45 ± 1 mL algal medium spiked
with acetone and algae
1 vessel, filled with 45 ± 1 mL algal medium spiked with
acetone without algae for analytical determination

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

The test material is instable in the study medium

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

The test material is instable in the study medium

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

The test material is instable in the study medium

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC0

Effect conc.:

> 0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

The test material is instable in the study medium

Basis for effect:

growth rate

Details on results:

The study was performed as a limit test using the concentration 2 mg/L nominal, because
the solubility of the test item in algal medium was determined as 1.94 mg/L. Because the
test item is not stable under test conditions and poorly soluble in test medium, acetone was
used to spike the concentration to be tested. The concentration of the solvent was 1000-fold
higher than the nominal concentration in the treatment. For the solvent control, only acetone
in a concentration of 1 mL/L was used. 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 particle counter. Growth rate μ and
the yield2 were determined from the cell number at the respective observation times.
Statistical evaluation was based on the comparison between treatment and solvent control.
No significant inhibition of algal growth was observed in the tested concentration compared
to the solvent control.
At the start and of the test and after 24 h, the content of the test item in the test solutions
was determined using HPLC-UV-determination. The concentration determined at the start
of the test was 76 % of the nominal concentration. After 24 hours, no more test item was
detectable. Therefore, the determination of the results was based on the geometric mean of
the measured concentrations (see OECD 201 (§ 39)). Because after 24 h no test item could
be measured in the test solution, ½ LOQ (0.01 mg/L) was used for calculation of the geometric
mean.
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).
Although the concentrations of the test material are low the algae were exposed to the hydrolytic
product as the substance undergo fast hydrolysis in the study medium.
The pH of the blank control should not fluctuate by more than 1.5 units. The change was 1.4
units in the blank control.
All validity criteria were met.
No observations were made which might cause doubts concerning the validity of the study
outcome. The result of the test can be considered valid.

Results with reference substance (positive control):

Parameter Value 95% confidence interval
72h ErC50 0.64 mg/L 0.58 – 0.71 mg/L
72h EyC50 0.32 mg/L 0.31 – 0.33 mg/L

Reported statistics and error estimates:

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.

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were met. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Conclusions:

The study was performed as a limit test using the concentration 2 mg/L nominal, because
the solubility of the test item in algal medium was determined as 1.94 mg/L. Because the
test item is not stable under test conditions and poorly soluble in test medium, acetone was
used to spike the concentration to be tested. The measured concentration was 0.12 mg/L. Nevertheless the tested organisms were exposed to the hydrolytic product as well.

Description of key information

The study was performed as a limit test using the concentration 2 mg/L nominal, because test item is not stable under test conditions and poorly soluble in test medium, acetone was used. The measured concentration was 0.12 mg/L. Nevertheless, the tested organisms were exposed to the hydrolytic product as well.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.12 mg/L

EC10 or NOEC for freshwater algae:

0.12 mg/L