[No public or meaningful name is available]

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 AUG 2020 - 20 DEC 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Guideline for Testing of Chemicals, National Institute of Environment Research, Chapter 3 Clause 2, 'Daphnia sp., Acute Immobilisation Test'

Version / remarks:

Notice No. 2019-23 (13 June, 2019)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

Adopted 13 April, 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC-MS/MS

Details on sampling:

- Concentrations: 100 mg/L (limit test)
- Sampling method:
Pretest (Stability test): The Stability test was conducted at the nominal concentrations of 12.5 and 100 mg/L. The test solutions were collected at 0 hr and 48 hr.
Main test: The test solutions for the control and the nominal concentrations of 100 mg/L were collected at 0 hr and 48 hr.
The test solutions of the nominal concentration of 100 mg/L were taken out from the middle layer of bottom. The prepared test solutions were filtered with 0.2 μm Top filter. The filtered test solution was analyzed using LC-MS/MS after 100-fold concentration using rotary evaporators.
- Sample storage conditions before analysis: No storage indicated

Vehicle:

no

Remarks:

a test solution was prepared using dilution water (M4 medium)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: During the preparation of test solution for the solubility test, it was observed that the test substance was completely separated from the dilution water while it was floated on the surface. Due to this phenomenon, the test substance was less dissolved to dilution water and the maximum solubility was determined to be < LOD/100. The test solution should be prepared by the same method that the maximum solubility has been achieved. However, the maximum solubility was not quantified. Therefore, the test solution was prepared by sonication treatment considering the efficiency of the test.
The limit test was conducted at the nominal concentration of 100 mg/L on the basis of the range-finding test result.
Measured 0.1 g (purity: 99.7 %) of the test substance was added to the 1 L of M4 medium in 1 L volume of glass tank to make 100 mg/L nominal concentration. And then the prepared test solution was sonicated for 30 minutes. The test solution was filtered using 0.2 µm bottle top filter after sonication to remove undissolved test substance and then it was used to the test. The test solution was prepared in the morning of exposure date.
- Controls: For the control, only the M4 medium was used.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): During the solubility test the test substance was completely separated from the dilution water while it was floated on the surface.
- Other relevant information: According to the method of OECD Test Guideline No. 202, M4 medium was used as culturing and dilution water in this study. Prior to use for the test, culturing and dilution water were aerated for 24 hours and pH, dissolved oxygen(DO), hardness and alkalinity of M4 medium were analyzed.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water fleas
- Justification for species other than prescribed by test guideline: Daphnia magna has been applied widely in Daphnia sp., acute immobilization test as a suitable experimental for toxicity testing. In addition, sufficient raw data has been accumulated and is available for interpretation and evaluation of study results.
- Age at study initiation (mean and range, SD): less than 24 hours
- Source: Risk Assessment Division, National Institute of Environmental Research
- Feeding during test: During the test duration, the daphnids used for the test were not fed.

ACCLIMATION
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: Cultures were fed daily with the green algae (Pseudokirchneriella subcapitata, provided by Neo&Biz) suspension once a day and the amount of feed was range of 1.0 mL (1×10^7 cells/mL) per culture (15 animal/1.5 L M4 medium).

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

248 mg/L (as CaCO3)

Test temperature:

20.0 - 20.1 ℃

pH:

7.71 - 8.02

Dissolved oxygen:

6.54 - 8.01 mg/L

Nominal and measured concentrations:

Nominal: 100 mg/L
Measured:

Details on test conditions:

TEST SYSTEM
- Test vessel: Test vessels were covered with transparent acrylic cover to reduce the loss of water due to evaporation and to avoid the entry of dust into the test solutions.
- Type (delete if not applicable): closed
- Volume of solution: 10 mL test solution per daphnid
- Aeration: The exposure period was 48 hours, the test solution was not aerated during test duration.
- Renewal rate of test solution (frequency/flow rate): limit test was performed using the semi-static method with the test solution replacement at 24 hours after exposure.
- No. of organisms per vessel: five daphnids per replicate
- No. of vessels per concentration (replicates): four replicates
- No. of vessels per control (replicates): four replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M4 medium
- Alkalinity: 48 mg/L (as CaCO3)

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light and 8-hour dark cycle (lighting on at 08:00 and off at 24:00)
- Light intensity: 644 - 689 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mobility, behaviour, appearance
Each test vessel was observed for immobility at 24 and 48 hours after exposure. The daphnids were considered to immobilized if they are not able to swim within 15 seconds after gentle agitation. In addition to immobility, any abnormal behavior and appearance were observed.

VEHICLE CONTROL PERFORMED: no vehicle used, for the control, only the M4 medium was used.

RANGE-FINDING STUDY
- Test concentrations: 12.5, 25, 50, 100 mg/L
- Results used to determine the conditions for the definitive study: The stability test was performed using the test solution of 12.5 and 100 mg/L nominal concentration, but the measured concentration was showed to be less than the LOD. Since the stability of the test substance in dilution water could not be confirmed, the limit test was performed using the semi-static method with the test solution replacement at 24 hours after exposure assuming the decreasing concentration.
Nominal concentrations of 12.5, 25, 50, 100 mg/L were used for the range finding study. There were no immobility and abnormal behavior in the control and all treatment groups.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other:

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mobility, behaviour, appearance

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mobility, behaviour, appearance

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mobility, behaviour, appearance

Details on results:

- Behavioural abnormalities: no
- Mortality of control: no toxic effects
- Other adverse effects control: no
- Immobilisation of control: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
(1) No immobilization and adverse symptoms were observed during 48 hours in the control group. Thus, the test was satisfied with criteria of test validity (less than 10%).
(2) The DO of the control and 100 mg/L test solution of nominal concentration were measured at the beginning(0 hr) and end (48 hr) of exposure, and range of the DO was 6.54 - 8.01 mg/L. Thus, the test was satisfied with criteria of test validity (above 3 mg/L).
- EC50 (24h): 1.73 mg/L (nominal)
- 95% confidence limits: 1.52 - 1.98 mg/L (nominal)

Reported statistics and error estimates:

No statistical treatment was done, since the limit test was carried out with only one test concentration. The EC50 values were expressed as above the test concentration.

Validity criteria fulfilled:

yes

Conclusions:

There were no immobility and abnormal behavior in the control and the 100 mg/L nominal concentration. In conclusion, in this 48-hour semi-static acute toxicity test on Daphnia magna the test item had no toxic effect at the saturation concentration following nominal loading of 100 mg test item/L; the EC50 and LOEC are higher than the solubility limit of the test item in the test medium, corresponding to a nominal concentration of > 100 mg/L.

Executive summary:

This study was performed to assess the acute toxicity to Daphnia magna according to OECD Guideline 202 under GLP. In the semi-static test, 20 young daphnia (5 animals in 4 replicates) were exposed to the test item in test media (M4 medium) for 48 hours.

The solubility test was conducted to confirm the maximum dissolved concentration of test substance in dilution water (M4 medium). The maximum solubility of the test substance was not analyzed. As determined by HPLC-MS/MS analysis, the concentration of the test substance dissolved in the M4 medium was lower than the limit of detection (LOD: 3.061 μg/L) and the test substance was considered to be dissolved in a very small amount (<0.03061 μg/L) in M4 medium.

The range-finding test was performed to determine the test concentration at 12.5, 25, 50, 100 mg/L of nominal concentration, and no toxic effects were observed at all concentrations. Therefore, the limit test was conducted at the control and nominal concentration of 100 mg/L. The stability test was performed using the test solution of 12.5 and 100 mg/L nominal concentration, but the measured concentration was showed to be less than the LOD. Since the stability of the test substance in dilution water could not be confirmed, the limit test was performed using the semi-static method with the test solution renewal at 24 hours after exposure assuming the decreasing concentration.

In the main study, no immobilisation or any abnormal behaviour of the test animals was observed in the control group and no immobilisation or any abnormal behavior of the test animals was observed at the test concentration of nominal 100 mg/L after 48 hours of exposure. The concentration of the test solution was considered to be lower than 0.03061 μg/L because the LOD was 3.061 μg/L and the test solution was concentrated 100-fold. Since the concentration of the test solution could not be analyzed, the median effective concentration (EC50) was expressed as less than the nominal concentration.

Accordingly, the 48-hour EC50 and the 48-h LOEC were determined to be > 100 mg test item/L, the 48-hour NOEC was determined to be ≥ 100 mg test item/L.

In conclusion, in this 48-hour semi-static acute toxicity test on Daphnia magna the test item had no toxic effect at the saturation concentration following nominal loading of 100 mg test item/L; the EC50 and LOEC are higher than the solubility limit of the test item in the test medium, corresponding to a nominal concentration of > 100 mg/L.

All validity criteria were met and, therefore, the study can be considered as valid.

Description of key information

Key, short-term toxicity to aquatic invertebrates, OECD 202, semi-static, limit-test, GLP: EC₅₀ (48h) > 100 mg/L nominal, analytical concentation <LOD (3.061 µg/L, since the test solution was 100x concentrated, i.e. <0.03061 µg/L) for Daphnia magna 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 100 mg/L

Additional information

This study was performed to assess the acute toxicity to Daphnia magna according to OECD Guideline 202 under GLP. In the semi-static test, 20 young daphnia (5 animals in 4 replicates) were exposed to the test item in test media (M4 medium) for 48 hours.

The solubility test was conducted to confirm the maximum dissolved concentration of test substance in dilution water (M4 medium). The maximum solubility of the test substance was not analyzed. As determined by HPLC-MS/MS analysis, the concentration of the test substance dissolved in the M4 medium was lower than the limit of detection (LOD: 3.061 μg/L) and the test substance was considered to be dissolved in a very small amount (<0.03061 μg/L) in M4 medium.

The range-finding test was performed to determine the test concentration at 12.5, 25, 50, 100 mg/L of nominal concentration, and no toxic effects were observed at all concentrations. Therefore, the limit test was conducted at the control and nominal concentration of 100 mg/L. The stability test was performed using the test solution of 12.5 and 100 mg/L nominal concentration, but the measured concentration was showed to be less than the LOD. Since the stability of the test substance in dilution water could not be confirmed, the limit test was performed using the semi-static method with the test solution renewal at 24 hours after exposure assuming the decreasing concentration.

In the main study, no immobilisation or any abnormal behaviour of the test animals was observed in the control group and no immobilisation or any abnormal behavior of the test animals was observed at the test concentration of nominal 100 mg/L after 48 hours of exposure. The concentration of the test solution was considered to be lower than 0.03061 μg/L because the LOD was 3.061 μg/L and the test solution was concentrated 100-fold. Since the concentration of the test solution could not be analyzed, the median effective concentration (EC50) was expressed as less than the nominal concentration.

Accordingly, the 48-hour EC50 and the 48-h LOEC were determined to be > 100 mg test item/L, the 48-hour NOEC was determined to be ≥ 100 mg test item/L.

In conclusion, in this 48-hour semi-static acute toxicity test on Daphnia magna the test item had no toxic effect at the saturation concentration following nominal loading of 100 mg test item/L; the EC50 and LOEC are higher than the solubility limit of the test item in the test medium, corresponding to a nominal concentration of > 100 mg/L.

All validity criteria were met and, therefore, the study can be considered as valid.