1-Ethyl-3-methylimidazolium Diethyl Phosphate

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:

2019-04-18 to 2019-05-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: The Guidelines for the Testing of Chemicals-Effects on Biotic Systems

Version / remarks:

2nd edition, Beijing: China Environment Press. 2013

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: The guidelines for the testing of chemicals

Version / remarks:

HJ/T 153-2004

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

Version of 2011

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1-Ethyl-3-methylimidazolium Diethyl Phosphate

Cas Number:

848641-69-0

Molecular formula:

C6H11N2 C4H10O4P

IUPAC Name:

1-Ethyl-3-methylimidazolium Diethyl Phosphate

Test material form:

liquid

Details on test material:

Batch No 187-9-113-Y
Expiry date: 2021-1-30

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 187-9-13-Y
- Expiration date of the lot/batch: 2021-1-30
- Purity test date: 2019-03-06


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep the container closed and in a dry, cool, well ventilated
area.
- Solubility and stability of the test substance in the test medium: good solublility; analysed concentration at test end was maintained above 80% of the initial measured concentration

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a stock liquid: 100 mg/L

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Test substance was analysed by UPLC-MS at 0 h and 72 h. The results showed that the concentration of 100 mg/L was between 93.0 mg/L and 98.6 mg/L during the 72 h test period.
In the limit Test, 10.0 mL water samples for the blank control group and 100 mg/L concentration groups were collected at 0 h, 24 h, 48 h and 72 h. The water sample is diluted 1000 times with
deionized water, then filtrated through 0.22μm membrane. Discard the first 1 mL, 1 mL of the remaining filtrate is analysed by UPLC-MS.

- Concentrations: limit test with 100 mg/L

Test solutions

Vehicle:

no

Details on test solutions:

Stock Solution of 100 mg/L for Limit Test was prepared by dissolving 0.5014 g of the test
substance into 500 mL steriled AAP medium.
Appropriate volumes of stock solution were added to glass flasks (150 mL) and diluted to 70 mL
with APP growth medium. Replication was not performed in the range-finding test. In the limit
Test, 7 replicates were used, 1 of which were used for the chemical analysis.

Test organisms

Test organisms (species):

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

Details on test organisms:

The freshwater algal species, Pseudokirchneriella subcapitata, was selected. It was obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology. For the limit test, Lot
No: A20190505P was used.
The algal medium is AAP. All nutrient salts of the medium were prepared as concentrated stock solutions and stored in the dark at 4 °C. Then the solution was sterilized by membrane filtration (mean pore diameter 0.45 μm) to obtain the fresh growth medium.

Preparation of Inoculum Culture
Pseudokirchneriella subcapitata originally obtained from Institute of Hydrobiology, Chinese
Academy of Science was cultured in AAP medium and transferred weekly. The initial
concentration was about 100 times smaller than that in the old culture.
2 ~ 4 days before the start of the test, a pre-culture was started by transferring 1.00 mL of stock
culture into conical flasks containing 100 mL sterile AAP medium. The pre-culture was
incubated on a shaker with 5000 Lux (±15%) continuous illumination at 23.0°C, and the rotor
speed was 100 (±10) rpm.
The Pseudokirchneriella subcapitata cultures were checked every day during pre-culture, no
deformed cells, fungus or other algae were observed, so these cultures met the test requirements

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

23°C to 23.6°C

pH:

see result table

Nominal and measured concentrations:

concentration of 100 mg/Ltest subtance was between 93.0 mg/L and 98.6 mg/L during the 72 h test period

Details on test conditions:

Range-finding Test
In the range-finding test, 3 widely-spaced concentrations of the test substance (1.00 mg/L, 10.0
mg/L and 100 mg/L) were used. A blank control group was run in parallel. Each test flask (total
volume: 150 mL) was filled with 70 mL of test solution, and the same biomass of exponentially
growing microalga pre-culture was inoculated to different concentrations of the test substance.
Flasks with these test cultures were capped with air-permeable stoppers and were then shaken and
randomly placed in the incubator.
In the incubator, the following conditions were maintained:
-Temperature: 23.0 °C ~23.6 °C;
-Light: continuous, uniform fluorescent illumination of cool-white type at 5617~5700 Lux;
-Rotor speed: 100 r/min;
Test duration was 72 hours. After administration of the test substance, the algal biomass in each
flask was determined by microscope at 24 h, 48 h and 72 h.


Limit Test
Based on the results of the range-finding test, test solutions of 100 mg/L were used in limit Test,
with 7 replicates for each test level as well as a blank control, and 1 vessels which were prepared
in order to determine the measured concentration. Water samples were collected at 0 h, 24 h, 48 h
and 72 h, which were analysed to determine the measured concentration.
Each test flask (Total volume: 150 mL) was filled with 70 mL of test solution, and the same
biomass of exponentially growing microalga pre-culture was inoculated to different
concentrations of the test substance. Flasks with these test cultures were capped with
air-permeable stoppers and were then shaken and randomly placed in the incubator.
In the incubator, the following conditions were maintained:
-Temperature: 23.0 °C to 23.6 °C;
-Light: continuous, uniform fluorescent illumination of cool-white type at 5523~5625 Lux;
-Rotor speed: 100 r/min;
Test duration was 72 hours. After the administration of the test substances, the algal biomass in
each flask was determined by microscope at 24 h, 48 h and 72 h. Microscopic observation was
also performed to verify a normal and healthy appearance of the inoculum culture and to observe
any abnormal appearance of the algae (as may be caused by the exposure to the test substance) at
the end of the test. Meanwhile, measurement of pH was carried out at the beginning and at the
end of the limit Test.

Reference substance (positive control):

yes

Remarks:

Under the same test conditions, K2Cr2O7 was used as the reference substance.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

98.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 98.6 mg/L

Nominal / measured:

meas. (initial)

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): no
- Effect concentrations exceeding solubility of substance in test medium: no

During the range-finding and Limit Test, algal cells in the control appeared normal. The algal
cells in the treatments no other abnormalities were detected.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 1.23 mg/L

Reported statistics and error estimates:

According to the statistical analysis of the test data by Independent Samples T-test (SPSS 17.0).,
the growth rates in treatment groups of 100 mg/L showed no significantly difference from that of
the blank control (p > 0.05). Thus it shows that under the valid test conditions, LOEC for
Pseudokirchneriella subcapitata was more than 98.6 mg/L (measured
concentration), and the NOEC for the test item was 98.6 mg/L (measured concentration).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under test conditions the test item is not toxic to algae up to a concentration of 100 mg/L.

Executive summary:

The Growth Inhibition Test to Alga (Pseudokirchneriella subcapitata) with the test substance was conducted according to “The guidelines for the testing of chemicals” (HJ/T 153-2004), “The Guidelines for the Testing of Chemicals-Effects on Biotic Systems” (the 2nd edition, Beijing: China Environment Press. 2013) and with reference to Procedure 201 of the “Guidelines for Testing of Chemicals” of the OECD: “Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (2011) etc.

During the whole test period, the pH values of the blank control mediums were between 7.30 and 7.97, and the pH of test mediums was between 7.19 and 7.82. The biomass in the control cultures increased exponentially with specific growth rates of 0.985 d-1, 1.09 d-1 and 1.26 d-1 for 0 ~ 1 d, 0 ~ 2 d and 0 ~ 3 d, respectively. Coefficients of variation of average specific growth rates for 0 ~ 1
d, 0 ~ 2 d and 0 ~ 3 d in replicate control cultures were 5.09%, 5.15% and 2.63 % (<7%), while the mean coefficient of variation for section-by-section specific growth rates (0 ~ 1 d, 1 ~ 2 d and 2 ~ 3 d for 72-hour tests) in the blank control cultures were 5.09%, 5.78% and 2.40% (<35%) respectively. Thus, the study met the acceptability criteria and was considered valid.
In the limit Test, the test solution was analysed by UPLC-MS at 0 h and
72 h. The results showed that the concentration of 100 mg/L test item was between 93.0 mg/L and 98.6 mg/L during the 72 h test period. Thus the test results were expressed with nominal concentration.

The results showed that under the valid test conditions, the 72 h-ErC50 of the test item to Pseudokirchneriella subcapitata was <100 mg/L, LOEC was >100 mg/L and the NOEC was 100 mg/L.