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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:
2008-02-22 - 2008-03-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: Acute Immobilization Test "Standard Pertaining to the Testing facility at Which Tests on New Chemical Substances, Etc.
Version / remarks:
” Pharmaceuticals and Medical Devices Agency Notification No. 1121003 on November 21, 2003, Manufacturing Industries Policy No. 3 on November 17, 2003, and the Ministry of Environment, the Environmental Policy Office Notification No. 031121004 (Final revision on April 1, 2005).
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
yes
Remarks:
dechlorinated water
Details on test solutions:
- Test concentration
As the results of preliminary examination (Report No. NCAS 07-223NG), a set concentration of 0.100, 0% immobilization in 1.00 mg/L area and 100% immobilization in the 10.0 mg/L area were observed. Thereby, tests were conducted in five concentrations (a common ratio of 2.0), which were 1.00, 2.00, 4.00, 8.00, and 16.0 mg/L in set concentration.

- Preparation of the test solution
50.0 mg of the test substance was weighed [and placed] in a 1000-mL measuring flask, added with dechlorinated water, and dissolved by an ultrasonic treatment (for ten minutes) to prepare a test stock solution 50.0 mg/L. 20.0, 40.0, 80.0, 160, and 320 mL of these test stock solutions were each sorted out in a 1000-mL volumetric flask and kept at a constant volume with dechlorinated water to prepare test solutions of 1.00, 2.00, 4.00, 8.00, and 16.0 mg/L. These test solutions were poured into four test containers 100 mL each. Dechlorinated water was used in the control area.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Generic name: Daphnia magna
- Scientific name: Daphnia magna
- Procured from: National Institute for Environmental Studies, Japan (the organisms procured on May 25, 2005 were subcultured in this office)
- Growth stage: Larvae born from grown crustacean 17-18 days old (less than 24 hours old, this test)
- Susceptibility: 48-hour EC50 background data according to the standard substance in this facility (potassium dichromate: reagent grade, Wako Pure Chemical Industries, Ltd.): 0.20-0.36 mg/L (n = 6, July 2005 to December 2007), immediate 48-hour EC50: 0.25 mg/L (Report no. NCAS 07-203, implemented in December 2007)

BREEDING CONDITIONS OF PARENT DAPHNIA TO OBTAIN TEST ORGANISMS
Only adult Daphnia were taken out from the subcultured Daphnia using a Komagome-type pipette and placed in a beaker, and the larvae born up to the next day were divided to separate beakers. These larvae (born on February 14-15, 2008) were bred as the parents of the test organisms. When the larvae were born, they were thinned at a rate of more than twice in a week. Only adult carrying eggs were used on the day before exposure, and the larvae born thereafter (less than 24 hours old) were used for the test (exposure started on March 4, 2008). During the breeding period, the death rate of parent Daphnia was 0%, and no dormant eggs, generation of male, and other abnormalities were observed.

- Water used for breeding: Dechlorinated water
- Breeding density: 25 daphnids (added at the start of breeding)/1 L of water used for breeding
- Breeding container: 1-L glass container
- Water temperature: 20.0ºC
- pH: 7.9-8.0
- Total hardness: 67-69 mg/L (in terms of hardness as CaCO3)
- Dissolved oxygen concentration: 8.7 mg/L (saturated concentration of 99% or higher)
- Illumination: Fluorescent lamp (715 Lux), 16 hours light/8 hours dark

FEEDING DURING BREEDING
- Feed: Unicellular green algae (Chlorella vulgaris, raw chlorella V12, Chlorella Industries)
- Feed volume: The feed was given at a rate of 0.1-0.2 mg-C per one Daphnia (organic carbon equivalent)/day. (The fish were fed by a peristaltic pump and a timer during holidays. Before the birth of larvae, the feed was given at the appropriate amount for the fish to consume.)
- Exchange of water used for breeding: More than once a week.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
69 mg/L (in terms of hardness as CaCO3)
Test temperature:
20.2ºC (Immersed in a constant temperature water tank installed in an immersion thermostat)
Water temperature inside the constant temperature water tank during the exposure period: 20.1-20.5ºC
pH:
7.8 - 7.9
Dissolved oxygen:
8.6 - 8.7 mg/L (98% or higher with respect to the saturated concentration)
Salinity:
not applicable
Conductivity:
not applicable
Nominal and measured concentrations:
1.00, 2.00, 4.00, 8.00, and 16.0 mg/L in set concentration
Details on test conditions:
TEST SYSTEM
- Test container: Glass beaker with 100-mL capacity
- Number of stations: Four container/concentration area
- Number of test organisms: 20 daphnids/concentration area (5 daphnids per one container x 4 containers)
- Feed: No feeding

TEST MEDIUM / WATER PARAMETERS
Dechlorinated water: We used tap water from Odawara city that has been treated with activated carbon to remove the residual chlorine, etc. and continuously and sufficiently aerated with an air pump. We confirmed that the quality of this water complied with the standard grade 3 fishery water (report no. NCAS 07-227NG on December 12, 2007). The total hardness before the experiment was measured and confirmed that it was in the range of 67-69 mg/L in terms of hardness as CaCO3

TEST CONCENTRATIONS
As the results of preliminary examination (Report No. NCAS 07-223NG), a set concentration of 0.100, 0% immobilization in 1.00 mg/L area and 100% immobilization in the 10.0 mg/L area were observed. Thereby, tests were conducted in five concentrations (a common ratio of 2.0), which were 1.00, 2.00, 4.00, 8.00, and 16.0 mg/L in set concentration.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
5.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
9 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Number of organisms immobilized and immobilization rate
The immobilization rate after 24-hour exposure was 0% in the control area, and in the 1.00, 2.00, and 4.00 mg/L area; 40% in the 8.00 mg/L area, and 100% in the 16.0 mg/L area. The immobilization rate 48 hours after exposure was 0% in the control area and the 1.00 mg/L area; 10% in the 2.00 mg/L area, 25% in the 4.00 mg/L area, 75% in the 8.00 mg/L area, and 100% in the 16.0 mg/L area. No Daphnia was found immobilized on the water surface in the control area.

Median immobilization concentration (EC50) and 95% confidence limit
EC50 at 24-hour exposure was 9.0 mg/L as calculated by the plotting method.
EC50 at 48-hour exposure was 5.2 mg/L as calculated by the Probit analysis, and its 95% confidence limit was in the range of 4.2-6.4 mg/L.

Results with reference substance (positive control):
Background data of 48-hour EC50: 0.20-0.36 mg/L
Reported statistics and error estimates:
Confirmation of the linearity of the calibration curve
The square (r2) of the correlation coefficient of the calibration curve was obtained, and the linearity of the calibration curve was confirmed.

Table 1: EC50and its 95% confidence limit throughout the 24- and 48-hour exposure

Exposure period

EC50(mg/L)

95% confidence limit (mg/L)

24 hours

9.0*

Not calculated due to plotting method

48 hours

5.2**

4.2-6.4

*Plotting method

**Probit analysis

Confirmation of the test substance

The spectrum described matched with the spectrum measured; therefore, the test substance was confirmed to be 2,5-xylenol.

Confirmation of stability of the test substance

The spectrum measured before the start of exposure matched [with the spectrum measured after the end of exposure], so the test substance was determined to be stable during the storage period.

Analytical method confirmation test of the concentration of the test substance in the test solution

The square of the correlation coefficient of the calibration curve was 0.9996 at the start of exposure and 1.0000 at the end of exposure. Thereby, it was determined that the linearity of the calibration curve was excellent.

In the confirmation of the repeatability, the concentration of the test substance in the test solution with 1.00 mg/L in set concentration was 1.00 mg/L in average (n = 3, 1.00, 0.989, and 1.01 mg/L), and the coefficient of variation was 1.05%. The concentration of the test substance in the test solution with 16.0 mg/L in set concentration was 15.9 mg/L in average (n = 3, 15.5, 15.7, and 16.6 mg/L), and the coefficient of variation was 3.68%. The coefficient of variation of the concentrations of the test substance in three stations was within 5%. No peak that could interfere with the quantification was observed at the detection position of the test substance in the HPLC chromatogram in the control area.

Based on these results, this analytical method was deemed reasonable as the method of measuring the concentration of the test substance in the test solution.

Evaluation of solubility of the test substance

As the result of confirmation by visual inspection that 100 mg of test substance was dissolved in 1 L of dechlorinated water, the solubility of the test substance was determined to be 100 mg/L or higher.

Concentration of the test substance in the test solution

The concentrations of the test substance in the test solution in each concentration area at the start of exposure were 1.00, 2.10, 3.96, 8.08, and 15.9 mg/L, which were 97.5-105% of the set concentration (1.00, 2.00, 4.00, 8.00, and 16.0 mg/L). The concentration of the test substance in the test solution in each concentration area 48 hours after exposure were 0.935, 1.95, 3.91, 7.91, and 15.8 mg/L, which were 93.5-98.9% of the set concentration.

Thereby, the variations in the concentration of the test substance at the start and the end of exposure with respect to the set concentration were less than ± 20%; therefore, the set concentration was used as the concentration of the test substance during the exposure period

Validity criteria fulfilled:
yes
Conclusions:
The median immobilisation concentration (EC50) after 48 hours was determined to be 5.2 mg/L.
Executive summary:

A GLP- acute immobilization test (static) of 2,5-xylenol on Daphnia magna in a 48-hour exposure was carried out in accordance with the acute immobilization test described in "Methods of Tests pertaining to New chemical Substances, Etc" and comparable to OECD Guideline 202.

20 daphnids per concentration area (5 daphnids/ container) were exposed to 2,5-xylenol at static conditions and at test concentrations of 1.00, 2.00, 4.00, 8.00 and 16.0 mg/L.

The concentrations of the test substance in the test solutions were 1.00, 2.00, 4.00, 8.00, 16.0 mg/L and in set concentrations were 1.00, 2.10, 3.96, 8.08, and 15.6 mg/L at the start of exposure and 0.935, 1.95, 3.91, 7.91, and 15.8 mg/L 48 hours after exposure. The proportion with respect to the set concentration was in the range of 97.5 - 105% at the start of exposure and 93.5 - 98.9% 48 hours after exposure, and the variation was less than ± 20% in either case. Thereby, the median immobilization concentration (EC50) was calculated using the set concentration as the concentration of the test substance during the exposure period.

EC50and its 95% confidence limit throughout the 24- and 48-hour exposure periods were shown below.

 

Exposure period

EC50(mg/L)

95% confidence limit (mg/L)

24 hours

9.0*

Not calculated due to plotting method

48 hours

5.2**

4.2-6.4

* Plotting method

** Probit analysis

The median immobilisation concentration (EC50) after 48 hours was determined to be 5.2 mg/L.

Description of key information

Acute Immobilization Test in Daphnia magna, 48 h, comparable to OECD Guideline 202, GLP, static, EC50 (48h) = 5.2 mg/L

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
5.2 mg/L

Additional information

A GLP- acute immobilization test (static) of 2,5-xylenol on Daphnia magna in a 48-hour exposure ( KS_Acute Immobilization Test in Daphnia magna_48 h _2008) was carried out in accordance with the acute immobilization test described in "Methods of Tests pertaining to New chemical Substances, Etc" and comparable to OECD Guideline 202.

20 daphnids per concentration area (5 daphnids/ container) were exposed to 2,5-xylenol at static conditions and at test concentrations of 1.00, 2.00, 4.00, 8.00 and 16.0 mg/L.

The concentrations of the test substance in the test solutions were 1.00, 2.00, 4.00, 8.00, 16.0 mg/L and in set concentrations were 1.00, 2.10, 3.96, 8.08, and 15.6 mg/L at the start of exposure and 0.935, 1.95, 3.91, 7.91, and 15.8 mg/L 48 hours after exposure. The proportion with respect to the set concentration was in the range of 97.5 - 105% at the start of exposure and 93.5 - 98.9% 48 hours after exposure, and the variation was less than ± 20% in either case. Thereby, the median immobilization concentration (EC50) was calculated using the set concentration as the concentration of the test substance during the exposure period.

EC50 and its 95% confidence limit throughout the 24- and 48-hour exposure periods were shown below.

 

Exposure period

EC50(mg/L)

95% confidence limit (mg/L)

24 hours

9.0*

Not calculated due to plotting method

48 hours

5.2**

4.2-6.4

* Plotting method

** Probit analysis

The median immobilisation concentration (EC50) after 48 hours was determined to be 5.2 mg/L.