[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:

December 21 2020 to December 24 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

copper(II) 3,3',3'',3'''-(29H,31H-phthalocyanine-2(or 3),9(or 10),16(or 17),23(or 24)-tetrayltetrasulfonyl)tetrapropane-1-sulfonate and its amides with (2-hydroxypropyl)amine, sodium salts

Molecular formula:

Cannot be adequately assigned

IUPAC Name:

copper(II) 3,3',3'',3'''-(29H,31H-phthalocyanine-2(or 3),9(or 10),16(or 17),23(or 24)-tetrayltetrasulfonyl)tetrapropane-1-sulfonate and its amides with (2-hydroxypropyl)amine, sodium salts

Test material form:

solid: particulate/powder

Details on test material:

Molecular formula: C50H52CuN10Na2O20S8 (main component)
Molecular weight: 1479.02 (main component)
Purity of test item: 93.1%
Impurity
Water: 6.5%
EDTA-4Na: 0.4%
Supplier: FUJIFILM Corporation
Lot number: M-002P
Storage conditions: Store in the dark at room temperature.

Specific details on test material used for the study:

Not specified

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Frequency of measurement: At the start and end of exposure
Sample for measurement: Another solution sampled separately from the preparation container
(at the start of exposure) The mixed solution taken out with equal volume of the test solution from the test vessels and the test solution from the vessel of analytical chemistry in each test level (at the end of exposure).
Removal of algae: Centrifugation (3 000 rpm, 10 minutes) (at the end of exposure).
Volume of sample: Approximately 200 mL (all test levels; at the start of exposure)
Approximately 122 mL [72 mL (from each the test vessel 24 mL) and approximately 50 mL (the vessel of analytical chemistry)] (all test levels; at the end of exposure).

Test solutions

Vehicle:

no

Details on test solutions:

The weighed test sample (0 .1 00 g) and medium of (931 mL) were mixed and stirred by a
magnetic stirrer until dissolving to prepare the stock solution of 100 mg/L. The test solution
was prepared in a preparation container by mixing required volume of the stock solution and
the medium and divided into each test vessel. In 100 mg/L level, the stock solution was divided into test vessel.

Test organisms

Test organisms (species):

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

Details on test organisms:

Reason for selection of species: Species recommended in the test guidelines
Source: American Type Culture Collection
Strain number: ATCC 22662
Supplied date: Supplied date
Subculture: Passage cultured under sterile conditions in this test facility Confirmation of reproducibility of test system An algae growth inhibition study of a reference substance with the
test organisms was periodically conducted. The latest data is shown below.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not specified

Test conditions

Hardness:

Not specified

Test temperature:

23.0 - 23.2 °C

pH:

7.8 - 7.9

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

100, 31.6, 10.0, 3.16 1.00 and 0.316 mg/L as nominal concentration (a geometric series with a factor of √10) The test concentrations and the geometric factor were decided from the results of the preliminary study. The results of the preliminary study are shown in Additional data.

Details on test conditions:

Type of test: Incubation with rotary shaking (approximately 100 rpm)
Exposure duration: 72 hours
Test concentrations: 100, 31.6, 10.0, 3.16 1.00 and 0.316 mg/L as nominal concentration (a geometric series with a factor of \{16) The test concentrations and the geometric factor were decided from the results of the preliminary study.
The results of the preliminary study are shown in Additional data.
Control: The medium without the test item
Replicate: 6 replicates/control (One additional test vessel for measuring the value of background was set.) 3 replicates/exposure level (One additional replicate for analytical chemistry of the test item in control and exposure levels was set, respectively.)
Volume of test solution: 300 mL/control (50 mL/test vessel) (One additional 50 mL for measuring the value of background was set.) 150 mL/exposure level (50 mL/test vessel) (One additional 50 mL for analytical chemistry of the test item in control and exposure levels was set, respectively.)
[This study was conducted half the usual volume (100 mL) considering influence of light path since the test sample was a coloured test chemical.]
Initial cell concentration: The algae were counted in pre-culture incubated under the same
conditions (but the light intensity was nominal90 ~mol·m-2 ·s-1) as the test for 3 days (from December 18 to December 21, 2020) as inoculums, and were added to test vessels to bring the initial cell concentration of 0. 7 5 x 10E4 cells/mL. The deformed and/or abnormal cells in the pre-culture used for the study was not present.
Operation: All operations were carried out under sterile conditions.
Temperature: 21-24 °C (not varied more than ±2 °C).
Light intensity: 115-125 J..Lmol·m-2·s-1 (the measured value of the arrangement
area of test vessel) in wavelength range of 400-700 nm Continuous illumination provided by alternative LED light to fluorescent lamp [This study was conducted near the upper limit (near 120
J..Lmol·m-2·s-1) of the test guideline considering influence of light since the test sample was a coloured test chemical.]

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Appearance of test solution
At the start of exposure, test solutions of all exposure levels were blue and clear
depending on the concentrations. At the end of exposure, the all appearance of test
solutions were bluish depending on the concentration, add that in 100 mg!L level was slightly
light green, that in 31.6 mg!L level was light green, those in 10.0 and 3.16 mg!L levels were
a little light green and those in the other exposure levels were green, due to the algal growth.
The solution of control was colorless and clear at the start of exposure, and it was green
at the end of exposure due to the algal growth.

Water quality and environmental conditions
The measured values of pH of the test solution are shown in Table 1, and culture
temperature and light intensity in the incubator are shown in Table 2.
The measured values of pH were 7.8-7.9. Culture temperatures in the incubator were 23.0-
23.20C and light intensities were 116-117 J..llllol·m-2·s-1.

Concentration of test item in test solution
The measured concentrations of test item in the test solutions at the start of exposure were
0.314-106 mg/L and those at the end of exposure were 0.297-111 mg/L, which were 99.3-
109% and 94.0-111% of the nominal concentration, respectively. The measured
concentrations of test item were maintained within the range of 80-120% of the nominal
concentrations.

ErC50
ErC50 of the test item based on the growth rate was > 100 mg/L.

Growth curve in each test level, cell observations and NOEC
The algal growth in 100 mg/L level was suppressed during exposure duration. The algal
growth in 31.6 mg/L level was better than that in 100 mg/L level, although growth inhibition
was found. The algal growth in 10.0 and 3.16 mg/L levels were logarithmic, although
growth inhibition was found. The algal growths in 1.00 and 0.316 mg/L levels were same
as control.
The conditions of cells in all exposure levels were same as the control. In the control, the
condition of cells was not abnormal.
On the growth rate, there were statistical differences in 100, 31.6, 10.0 and 3.16 mg/L
exposure levels. By the results in statistical analysis and cell observation showed above,
NOEC based on the growth rate was estimated at 1.00 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

As a result, ErC50 was > 100 mg/L and NOEC was 1.00 mg/L.

Executive summary:

The objective of this study is to determine the median effective concentration (EC50) and no observed effect concentration (NOEC) of 0-72-hour by conducting an algae growth inhibition study of Dye-2019 in Pseudoldrchneriella subcapitata. The 72 hour algal growth inhibition study carried out in accordance with OECD test guideline 201, in compliance with GLP.

This study was conducted in order to estimate the effect on the test organisms under the solubility of test item in medium.

Since the test sample was a coloured test chemical, this study was conducted considering influence of the light with reference to OECD Guidance Document No. 23 as well.

This study was conducted near the upper limit (near 120 J..Lmol·m-2·s-1) of the test guideline considering influence of light. In addition, that was conducted half (50 mL) the usual volume (100 mL) considering influence of light path, further the water depth was made shallow using 500 mL erlenmeyer flask.

The measured concentrations of the test item in the test solutions were within the range of ±20% of the nominal concentration.

As a result, ErCso was > 100 mg/L and NOEC was 1.00 mg/L.

The environmental conditions were within the suitable range. Therefore, it is concluded that this study complied with the applied test guidelines.