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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017/11/23 — 2018/02/07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Chemical name: DS-PHYTOSPHINGOSINE
Lot No.: PYFF01
Date received: 2017. 11. 10.
Amount: 75.18 g(including container)
Manufacturing date: 2017. 06. 01.
Expiration date: 2019. 05. 31.
Analytical monitoring:
yes
Details on sampling:
Quantitative analysis of the test substance were performed in each replicate at the beginning(0 hr), at the end of the test(48 hrs) and just before and after renewal of the test solution after 24h.
Vehicle:
no
Details on test solutions:
Preparation of stock solution
The required amount of the test substance were weighed (0.1013 g definitive study) and mixed with breeding water. This suspension was mixed using a sonicator (HWASHINTECH, Korea) for 20 min. The suspension were filtered through a 0.22 gm PVDF membrane filter (Hyundai Micro Co., LTD., Korea). This 100 mg/L stock solution were used for the dilution of each treatment groups.

Preparation of test solution
For the definitive study, the stock solution of 100 mg/L was prepared and the test solutions of 6.25, 12.5, 25.0, 50.0 and 100 mg/L was prepared by diluting the stock solution with breeding water. 100 mL per dish were prepared for each treatment group and only breeding water was used for the control group.
All test dishes of the treatment groups and control group were observed for the state of test solutions(transparency, turbidity, precipitation, floating, flocculation, etc.) prior to initiation of exposure and at 24(before and after renewal of test solution) and 48 hrs after exposure.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM Supplier :
- Common name: Daphnia sp.(Daphnia magna)
- Source: In the Daphnia sp. breeding room of Centralbio Co., Ltd.TEST ORGANISM

Breeding method of Daphnia sp.:
Young Daphnia sp. of less than 24 hrs old were obtained from the mature Daphnia sp. of more than 14 days old and giving birth to over three generations and were transferred to new culture beakers and bred for 28 days. The culture beakers were maintained at 18-22 °C in a water bath on a cycle of 16 hrs of continuous artificial light and 8 hrs of continuous darkness. Daphnia sp. were fed daily with a suspension of Chlorella sp.(1.0 x 104 — 105 cells/mL/20 Daphnia sp.). During the culture period, culture beakers holding Daphnia sp. were observed for general symptoms(behavior, antennae movement and appearance), neonate production and ephippia once daily except on weekend or holiday(at least once every three days). Young Daphnia sp. of less than 24 hrs old obtained from the mature Daphnia sp. of more than 14 days old and giving birth to over three generations were used for this study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Daphnid(Daphnia magna) was exposed once every six months to a reference substance(Potassium dichromate, Sigma-Aldrich) for 24 and 48 hrs and EC50 was determined. The result(EC50) of the positive control study was within the permissible range of historical data(mean ± SD) of Centralbio Co., Ltd. Exposure conditions of the positive control study were identical to the conditions of the definitive study.
Hardness:
150 — 155 mg CaCO3/L
Test temperature:
19.4 — 20.6 °C
pH:
7.81 — 8.05
Dissolved oxygen:
6.88 — 9.45 mgO2/L (converted to air saturation value: 75.2 — 103.2 %)
Nominal and measured concentrations:
Nominal concentrations: 6.25, 12.5, 25.0, 50.0, 1000 mg/L
Measured concentrations: 0.0192, 0.0317, 0.0913, 0.188, 0.498 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: dishes(080x40H(mm))
- Fill volume: 100 mL
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 2
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Salinity:
- Culture medium different from test medium:
- Intervals of water quality measurement: The water temperature, dissolved oxygen concentration and pH of the test solutions were measured from one test dish each for the treatment groups and control group
prior to initiation of exposure and at 24(before and after renewal of test solution) and 48 hrs after exposure. The water temperature in the water bath were also recorded.

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- General symptoms: behavior, antennae movement and appearance in the treatment groups and control group at 24 and 48 hrs after exposure
- Immobilisation: number of Daphnia sp. at immobilisation (mobile: Daphnia sp. move independently under water using the second antenna; immobilisation: unable to swim even though some organs (second antennae, posterior abdomen), do not move for approximately 15 seconds after dishes are gently shaken).

RANGE-FINDING STUDY
- Test concentrations: 0.100, 1.00, 10.0 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.131 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CI: 0.106 - 0.161
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.032 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Range-finding study
In the range finding study, all the daphnids in the test solution of 100 mg/L were daed and another test concentrations(0.1, 1 and 10 mg/L) did not show immobilisation or abnormal symptoms. Therefore, the highest concentration of the test was set at 100 mg/L and the following four concentrations were set as 6.25, 12.5, 25.0 and 50.0 mg/L as an azeotropic ratio of 2.00. In addition, control group was set.

Definite study
Cumulative number of Immobilisation Daphnia sp. and general symptoms:
During the exposure period, decrease of antennae movement and lay down on the side was evident in the 25.0 mg/L treatment groups; decrease of antennae movement, and death was evident in the 50.0 mg/L treatment group; death was found in the 100 mg/L treatment groups. However, no immobilisation or abnormal signs were evident in the control group or in the 6.25 and 12.5mg/L treatment group throughout the course of the study.

EC50, NOEC and EC100:
During the exposure period, the EC50 was determined to be > 0.498 mg/L at 24 hours after exposure and 0.131 mg/L(95 % confidence limits: 0.106 — 0.161 mg/L) at 48 hours after exposure.
During the exposure period, the highest concentrations causing no immobility were determined to be 0.188 mg/L at 24 hours after exposure and 0.0317 mg/L at 48 hours after exposure, and the lowest concentrations causing 100 % immobility were determined to be 0.0317 at 24 hours and 0.498 mg/L at 48 hours after exposure. Observed immobilisation of Daphnia magna followed a dose-response relationship.


State of test solutions
The test solutions in the control group or in the 6.25, 12.5, 25.0, 50.0 and 100 mg/L treatment groups were transparent prior to initiation of exposure and during the exposure period.
Results with reference substance (positive control):
The result(EC50) of the positive control study was within the permissible range of historical data(mean ± SD) of Centralbio Co., Ltd.: 48h-EC50 = 0.865 mg/L (95% CI: 0.768 ~ 0.974) (study period: 2017/10/18 ~ 2017/10/20)
)
Reported statistics and error estimates:
The median effective concentration(EC5o)
Statistical analysis was performed using the U.S. EPA authorized program. For the treatment groups and control group, a concentration-immobility curve was generated and the EC50, and its 95% confidence limits for immobility at 48 hours after exposure were analyzed using Probit. For the treatment groups and control group at 24 hours, the immobility was less than 50 %(actual measurement: 0 %), thus statistical analysis was not performed. Therefore, the EC50 at 24 hrs after exposure was determined to be > the highest concentration in this study.

Cumulative immobilisation data for Daphnia magna during the definitive study

Desired
nominal
concentration
(mg/L)

Mean measured
concentration
(mg/L)

Number of

Cumulative number of organisms
immobilised (Immobility, %)

organisms
tested

24 hrs

48 hrs

Control

ND*

20

0(0)

0(0)

6.25

0.0192

20

0(0)

0(0)

12.5

0.0317

20

0(0)

0(0)

25.0

0.0913

20

0(0)

4(20)

50.0

0.188

20

0(0)

16(80)

100

0.498

20

9(45)

20(100)

*: Not detected

General symptoms of Daphnia magna during the definitive study

Desired
nominal
concentration
(mg/L)

Mean measured
concentration
(mg/L)

Number of
organisms

General symptoms(Number of Daphnia magna)

tested

24 hrs

48 hrs

Control

ND*

20

N(20)

N(20)

6.25

0.0192

20

N(20)

N(20)

12.5

0.0317

20

N(20)

N(20)

25.0

0.0913

20

N(15)
g(5)

N(11)
g(5)

e(4)

50.0

0.188

20

N(2)
g(18)

g(4)
f(16)

100

0.498

20

g(11)
e(9)

f(20)

N: Normal

g: Decrease of antennae movement

e: Lay down on the side(symptom of immobilisation)

f: Death(symptom of immobilisation)

*: Not applicable

Validity criteria fulfilled:
yes
Conclusions:
Derived effect concentration: 48h-EC50 (Daphnia magna, immobility) = 0.131 mg/L.
Executive summary:

An Acute Immobilisation Test of Phytosphingosine in (Daphnia magna was performed according to OECD 202 (2004) and in compliance with GLP. Daphnia magna was exposed to five test concentrations (6.25, 12.5, 25.0, 50.0 and 100 mg/L) for 48 hours under semi-static test conditions. A control was run in parallel. All test concentrations were analytically verified. Measured values of the test solutions prepared at 6.25, 12.5, 25.0, 50.0 and 100 mg/L were 0.0192, 0.0317, 0.0913, 0.188 and 0.498 mg/L, respectively. These values were out of a range of 80 to 120 % compared with the desired nominal concentrations. Therefore, all test results were based on the mean measured concentrations.

During the exposure period, the EC50 was determined to be > 0.498 mg/L at 24 hours after exposure and 0.131 mg/L(95 % confidence limits: 0.106 — 0.161 mg/L) at 48 hours after exposure. During the exposure period, the highest concentrations causing no immobility were determined to be 0.188 mg/L at 24 hours after exposure and 0.0317 mg/L at 48 hours after exposure, and the lowest concentrations causing 100 % immobility were determined to be 0.0317 at 24 hours and 0.498 mg/L at 48 hours after exposure. Observed immobilisation of Daphnia magna followed a dose-response relationship.

The validity criteria of the guideline were fulfilled and results considered reliable and adequate for the aquatic hazard assessment.

Description of key information

48h-EC50 (Daphnia magna, immobility) = 0.131 mg/L (mean measured, OECD 201, 2018)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.131 mg/L

Additional information

A reliable key study was available investigating acute immobilisation of Phytosphingosine in Daphnia magna. The study was performed according to OECD 202 (2004) and in compliance with GLP. Daphnia magna was exposed to five test concentrations (6.25, 12.5, 25.0, 50.0 and 100 mg/L) for 48 hours under semi-static test conditions. A control was run in parallel. All test concentrations were analytically verified.

During the exposure period, the EC50 was determined to be > 0.498 mg/L at 24 hours after exposure and 0.131 mg/L (95 % confidence limits: 0.106 - 0.161 mg/L) at 48 hours after exposure.

During the exposure period, the highest concentrations causing no immobility were determined to be 0.188 mg/L at 24 hours after exposure and 0.0317 mg/L at 48 hours after exposure, and the lowest concentrations causing 100 % immobility were determined to be 0.0317 at 24 hours and 0.498 mg/L at 48 hours after exposure (based on mean measured concentrations). Observed immobilisation of Daphnia magna followed a dose-response relationship.

The validity criteria of the guideline were fulfilled and results considered reliable and adequate for the aquatic hazard assessment.