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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-02-22 - 2008-03-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: fish acute toxicity test described in the “Methods of Tests pertaining to New Chemical Substances," Japan
Version / remarks:
stipulated in the Notification by the Director of Pharmaceutical Affairs and Food Sanitation of the Ministry of Health, Labor and Welfare; Director for Manufacturing Industries Policy of the Ministry of Economy, Trade, and Industry; and Director of Environmental Policy of the Ministry of the Environment (Pharmaceutical Affairs and Food Sanitation Notification No. 1121002 dated November 21, 2003, Manufacturing Industries Policy No. 2 on November 13, 2003, and the Ministry of Environment, the Environmental Policy Office Notification No. 031121002), final revision dated November 20, 2006.
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
yes
Remarks:
dechlorinated water
Details on test solutions:
- Test concentration
Based on the results obtained from the preliminary study (Report No. NCAS 07-223NG), 0% and 100% of death were confirmed in the areas 1.00 and 10.0 mg/L in set concentration, respectively. Thereby, the test was conducted in five concentrations (common ratio of 2.0), which were 1.00, 2.00, 4.00, 8.00, and 16.0 mg/L in set concentration.

- Confirmation of solubility and preparation of the test solution
100 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 15 minutes), and kept at a constant volume with dechlorinated water to confirm solubility. 30.0, 60.0, 120, 240, and 480 mL of these test solutions were each sorted out in a 3-L beaker to prepare test solutions of 1.00, 2.00, 4.00, 8.00, and 16.0 mg/L with 3.0 L of dechlorinated water. Dechlorinated water was used in the control area. The state of the test solutions (appearance, etc.) was recorded

Test organisms (species):
Oryzias latipes
Details on test organisms:
TEST ORGANISM
- Generic name: Japanese rice fish
- Scientific name: Oryzias Latipes
- Procured from: the Fish Hunan (procured on January 9, 2008)
- Birth month: May 2007
- Average length and weight: 3.3 cm (3.0-3.4 cm), 0.23 g (0.21-0.27 g), the average values of ten fishes randomly captured at the start of exposure (measured on March 3, 2008)
- Susceptibility: 96-hour LC50 according to the standard substance (as the anhydrate of copper sulfate (II) (molecular weight 159.61): reagent grade, Wako Pure Chemical Industries, Ltd.): 1.2 mg/L (95% confidence limit 0.7-12 mg/L, February 2008).

ACCLIMATION
The fish were acclimated for ten days (February 22-March 3, 2008) under the following conditions. No abnormalities were found in the fish during the acclimatization period. The death rate during the acclimatization period was 2.2%.
- Water used for breeding: Dechlorinated water
- Breeding container: 100-L glass water tank
- Water temperature: 22.8-23.0ºC
- Dissolved oxygen concentration: 86-88%
- pH: 7.3-7.4
- Illumination: Fluorescent lamp, 16 hours light/8 hours dark

FEEDING DURING TEST
- Feed: Feed for Japanese rice fish (Tetra Japan)
- Feeding volume: Feed was given at a ratio of 1.5% equivalent of the fish weight once a day. Fasting was carried out from about 54 hours before the start of exposure.
- Feed method: Static method
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
51-53 mg/L (in terms of hardness as CaCO3)
Test temperature:
22.8 - 23.0ºC (immersed in the constant temperature bath equipped with an immersion thermostat)
Water temperature of the constant temperature bath during the exposure period: 22.7 - 23.1ºC
pH:
7.3 - 7.7
Dissolved oxygen:
76 - 100% (6.4 - 8.4 mg/L)
Salinity:
not applicable
Conductivity:
not applicable
Nominal and measured concentrations:
1.00, 2.00, 4.00, 8.00, and 16.0 mg/L and control area
Details on test conditions:
TEST SYSTEM
- Test vessel: 3-L glass beaker (no sealing)
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 3 L/test water tank
- Aeration: None
- Renewal rate of test solution (frequency/flow rate): Semi-static method (released type, 24-hour water exchange)
- No. of organisms per vessel: Ten fish/concentration area
- No. of vessels per concentration (replicates): One test water tank for one concentration area
- No. of vessels per control (replicates): one

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 hardness during the exposure period was measured and confirmed that it was in the range of 51-53 mg/L in terms of hardness as CaCO3

TEST CONCENTRATIONS
Based on the results obtained from the preliminary study (Report No. NCAS 07-223NG), 0% and 100% of death were confirmed in the areas 1.00 and 10.0 mg/L in set concentration, respectively. Thereby, the test was conducted in five concentrations (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:
Copper (II) sulfate pentahydrate
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
5.7 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
5.9 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
6.6 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
11 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Concentration of the test substance and death rate
No death was observed in the control area and the 1.00-2.00 mg/L concentration areas during all exposure periods. 10% of death was observed 96 hours later in the 4.00 mg/L concentration area, and 10, 70, 90, and 90% of death were observed 24, 48, 72, and 96 hours later, respectively, in the 8.00 mg/L concentration area. Furthermore, although 0% death was observed 3 hours later in the 16.0 mg/L concentration area, 30% death was observed 6 hours later and 100% death was observed 24 hours later. Thereby, the highest concentration producing 0 percent death was 4.00 mg/L 24, 48, and 72 hours later; and 2.00 mg/L 96 hours later. The lowest concentration producing 100 percent deaths was 16.0 mg/L 24-96 hours later.

Median lethal concentration (LC50)
The death rate (%) was calculated from the number of death and the number of test specimens (ten fish) 96 hours after exposure in each concentration range, and the results of calculating the median lethal concentration (95% confidence limit) by the Probit analysis was 5.7 mg/L (4.4-7.2 mg/L). Some [solutions] had minimal concentration of death 24, 48, and 72 hours after exposure, so it was not possible to calculate using the Probit analysis; therefore, the plotting method was used, and the results were 11, 6.6, and 5.9 mg/L, respectively.

Observation results of the test fish
No symptoms of abnormal mobility, etc. were observed in the concentration area of 1.00 mg/L or lower. Abnormal mobility and death were observed 96 hours later in the 4.00 mg/L concentration area. Abnormal mobility or death were observed in some specimens 6 hours later in the 8.00 mg/L concentration area. All specimens died 24 hours later in the 16.00 mg/L concentration area. No turbidity and precipitation were observed in the test solution during the exposure period, and the color tone was colorless and transparent in all concentration areas.
Results with reference substance (positive control):
- Results with reference substance: valid
- LC50: 0.75 mg/L

Table 1: median lethal concentration (LC50), the highest concentration producing 0 percent death, and the lowest concentration producing 100 percent deaths 24, 48, 72, and 96 hours after exposure

Exposure period  LC50* (95% confidence limit)
(mg/L)
Highest concentration producing 0 percent death
(mg/L)
Lowest concentration producing 100 percent deaths
(mg/L)

24 hours 11* 4.00 16.0
48 hours 6.6* 4.00 16.0
72 hours 5.9* 4.00 16.0
96 hours 5.7** (4.4 - 7.2) 2.00 16.0

*: Obtained by the plotting method.

**: Obtained by Probit analysis.

Confirmation of the test substance

The spectrum described matched with the spectrum measured; therefore, the test substance was determined 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 storage.

 

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

HPLC chromatogram of the control area shows at the start of the exposure and the confirmation of repeatability (16.0 mg/L test solution), and the standard solution (100 mg/L).

The square of the correlation coefficient of the calibration curve was 0.9996 or higher at the time of measuring the concentration of the test substance during the exposure period and the analytical method confirmation test. Thereby, it was determined that the linearity of the calibration curve was excellent.

In the confirmation of repeatability, the average concentration of the test substance in the test solution with 1.00 mg/L in concentration was 0.901 mg/L (n = 3, 0.896, 0.913, and 0.894 mg/L), and the coefficient of variation was 1.16%. The average concentration of the test substance in the test solution with 16.0 mg/L in concentration was 16.3 mg/L (n = 3, 16.0, 16.5, and 16.3 mg/L), and the coefficient of variation was 1.55%. 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 dissolving 100 mg of test substance 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 variations of the concentrations of the test substance measured with respect to the set concentration were all less than ± 20%; therefore, the average measured concentration was not calculated, and the set concentration was used as the concentration of the test substance during the exposure period.

Validity criteria fulfilled:
yes
Conclusions:
The median lethal concentration (LC50) after 96 hours was determined to be 5.7 mg/L.
Executive summary:

A GLP compliant 96-hour acute toxicity test of 2,5-xylenol on Oryzias latipes has been conducted in accordance with the acute toxicity test described in "Methods of Tests pertaining to New Chemical Substances, Etc" and comparable to OECD Guideline 203.

Ten fish per test concentration (10.00, 2.00. 4.00. 8.00 and 16.0 mg/L) were evaluated in a semi-static 96 -hour acute toxicity test. Mortalities as well as symptoms of abnormal mobility has been evaluated. No symptoms of abnormal mobility, etc. were observed in the concentration area of 1.00 mg/L or lower. Abnormal mobility and death were observed 96 hours later in the 4.00 mg/L concentration area. abnormal mobility or death were observed in some specimens 6 hours later in the 8.00 mg/L concentration area. All specimens dies 24 hours later in the 16.00 mg/L concentration area.

Since the variations to the set concentration of the measured concentration of the test substance were all less than ± 20%, the set concentration was used as the concentration of the test substance in the test solution. The median lethal concentration (LC50), the highest concentration producing 0 percent death and the lowest concentration producing 100 percent deaths 24, 48, 72, and 96 hours after exposure were shown below.

Exposure period  LC50* (95% confidence limit)
(mg/L)
Highest concentration producing 0 percent death
(mg/L)
Lowest concentration producing 100 percent deaths
(mg/L)

24 hours 11* 4.00 16.0
48 hours 6.6* 4.00 16.0
72 hours 5.9* 4.00 16.0
96 hours 5.7** (4.4 - 7.2) 2.00 16.0

* Obtained by the plotting method.

** Obtained by Probit analysis

The median lethal concentration (LC50) after 96 hours was determined to be 5.7 mg/L.

Description of key information

Acute toxicity to freshwater fish, comparable to OECD 203, GLP, Oryzias latipes, 96 h semi-static: LC50 (96h) = 5.7 mg/L

Key value for chemical safety assessment

LC50 for freshwater fish:
5.7 mg/L

Additional information

A GLP compliant 96-hour acute toxicity test of 2,5-xylenol on Oryzias latipes (KS_Acute toxicity to freshwater fish_Oryzias latipes_96 h

_2008) has been conducted in accordance with the acute toxicity test described in "Methods of Tests pertaining to New Chemical Substances, Etc" and comparable to OECD Guideline 203.

Ten fish per test concentration (10.00, 2.00. 4.00. 8.00 and 16.0 mg/L) were evaluated in a semi-static 96-hour acute toxicity test. Mortalities as well as symptoms of abnormal mobility has been evaluated. No symptoms of abnormal mobility, etc. were observed in the concentration area of 1.00 mg/L or lower. Abnormal mobility and death were observed 96 hours later in the 4.00 mg/L concentration area. Abnormal mobility or death were observed in some specimens 6 hours later in the 8.00 mg/L concentration area. All specimens died 24 hours later in the 16.00 mg/L concentration area.

Since the variations to the set concentration of the measured concentration of the test substance were all less than ± 20%, the set concentration was used as the concentration of the test substance in the test solution. The median lethal concentration (LC50), the highest concentration producing 0 percent death and the lowest concentration producing 100 percent deaths 24, 48, 72, and 96 hours after exposure were shown below.

Exposure period  LC50* (95% confidence limit)
(mg/L)
Highest concentration producing 0 percent death
(mg/L)
Lowest concentration producing 100 percent deaths
(mg/L)

24 hours 11* 4.00 16.0
48 hours 6.6* 4.00 16.0
72 hours 5.9* 4.00 16.0
96 hours 5.7** (4.4 - 7.2) 2.00 16.0

* Obtained by the plotting method.

** Obtained by Probit analysis

The median lethal concentration (LC50) after 96 hours was determined to be 5.7 mg/L.