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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-07-31 - 2008-09-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: Growth Inhibition Test in Algea in accordance with the “Standard Pertaining to the Testing facility at Which Tests on New Chemical Substances, Etc.
Version / remarks:
stipulated in 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 July 4, 2008).
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
yes
Remarks:
OECD culture medium
Details on test solutions:
- Test concentration
Based on the results obtained from the preliminary study (scheduled to be reported in Report No. NCAS 08-072NG), the growth inhibition rates based on the growth rate for 0-72 hours were 14.8% in 12.5 mg/L, 37.2% in 25.0 mg/L, and 88.4% in 50.0 mg/L. Thereby, the test was conducted in five concentrations (common ratio of 2.0), which were 5.00, 10.0, 20.0, 40.0, and 80.0 mg/L in set concentration.

Preparation of the test solution
The test substance was weighed in the amount of 50.0, 40.0, 20.0, and 10.0 mg, each was placed in a 500-mL volumetric flask, added with an OECD culture medium in the amount of about 450 mL, and dissolved by an ultrasonic treatment (within 30 minutes). After it was left to cool for about 30 minutes in a clean bench, it was kept at a constant volume with an OECD culture medium to prepare 100 mg/L of test stock solution, as well as 80.0, 40.0, and 20.0 mg/L of test solutions.
50.0 and 25.0 mL of test stock solution 100 mg/L were each sorted in a 500-mL volumetric flask and kept a constant volume in OECD culture medium to prepare 10.0 and 5.00 mg/L test solution.
Three test containers for each concentration area were used, and each container was added with 100 mL of the test solution. Six test containers were used for the control area, and each container was added with 100 mL of OECD culture medium without containing the test substance.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Classification: Unicellular green algae
- Scientific name: Pseudokirchneriella subcapitata (Selenastrum capricornutum)
- Procured from: American Type Culture Collection (ATCC) Manassas, VA 20108, USA Summit Pharmaceuticals International (procured on August 2, 2001)
- Strain name: ATCC 22662
- Susceptibility: As the results of conducting a growth inhibitory test on the standard substance (potassium dichromate: reagent grade, Wako Pure Chemical Industries, Ltd.) during the test period, 72-hour EC50 was 0.84 mg/L (velocity method), and it was determined to be no problem as the value of susceptibility
The background data of 72-hour ErC50 according to the standard substance (potassium dichromate) in this facility (velocity method) (data of algae obtained on October 13, 2005): 0.66-0.82 mg/L (n = 4, December 2006-January 2008)

ACCLIMATION
- Preculture conditions: The algae supplied for this test were allowed to grow throughout their exponential period cultured for three days before the start of exposure under test conditions described before. At the end of the preculture, the appearance of abnormal cells such as deformity was not observed.
Since the preculture was started at a cell concentration of about ten times (50005 cells/mL, calculation value) higher than the standard of this test, the dry weight of the test solution in the control area after the end of exposure was measured, and along with the results of the growth curve and the increase rate in the control area of this test and the susceptibility test, we considered these may possibly affect the test results.
- Period: 3 days
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not specified
Test temperature:
22.4-23.0ºC (temperature inside the device, tolerance: 23 ± 2ºC)
pH:
7.8-8.0 (pH adjustment of the test solution was not performed)
Dissolved oxygen:
not specified
Salinity:
not applicable
Conductivity:
not applicable
Nominal and measured concentrations:
5.00, 10.0, 20.0, 40.0, and 80.0 mg/L
Details on test conditions:
TEST SYSTEM
- Culture method: Shake culture (100 rpm)
- Exposure period: 72 hours
- Test solution volume: 100 mL per one container
- Test container: 300-mL glass Erlenmeyer flask with an aluminum cap
- Number of stations: 3 containers/concentration area (6 containers in the control area)
- Initial cell concentration: 5005 cells/mL (calculated value)
- Test temperature: 22.4-23.0ºC (temperature inside the device, tolerance: 23 ± 2ºC)
- Illumination: Continuous luminance at 5200-5800 Lux, 65-71 μE/m2/s (in the vicinity of the level of the flask liquid (tolerance: fluctuation was [controlled] to be within ± 20%)


TEST MEDIUM / WATER PARAMETERS
OECD culture medium


TEST CONCENTRATIONS
Based on the results obtained from the preliminary study (scheduled to be reported in Report No. NCAS 08-072NG), the growth inhibition rates based on the growth rate for 0-72 hours were 14.8% in 12.5 mg/L, 37.2% in 25.0 mg/L, and 88.4% in 50.0 mg/L. Thereby, the test was conducted in five concentrations (common ratio of 2.0), which were 5.00, 10.0, 20.0, 40.0, and 80.0 mg/L in set concentration.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
29 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Growth curve and growth rate
The cell concentration in the control area increased 79 fold in average in 72- hour culture, exceeding 16 fold specified in the test plan.
The average value of the coefficient of variation of the daily growth rate (μ1, μ2, and μ3) in the control area was 31.6%. The coefficient of variation between each repeatability in the growth rate (μ4) at 0-72 hours in the control area was 2.35%. Each value was within the range specified in the test plan. Based on the results above, the algae in the control area showed normal growth under the test conditions.

- 50% growth inhibitory concentration (ErC50) and maximum no observed effect concentration (NOEC)
EC50 (0-72 h) was 29 mg/L, and its 95% confidence limit was in the range of 26-32 mg/L. NOEC (0-72 h) was determined to be 5.00 mg/L
Results with reference substance (positive control):
- Results with reference substance valid
- Background data of 72-hour ErC50: 0.84
Reported statistics and error estimates:
The squares of the correlation coefficient of the calibration curve were 1.0000 (at the start of exposure) and 0.9998 (at the end of exposure); thereby, the linearity of the calibration curve was confirmed

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 storage.

Evaluation of solubility of the test substance

It was confirmed that 10.02 mg of test substance was dissolved in 100 mL of OECD culture medium. The solubility of the test substance with respect to the OECD culture medium was determined to be 100 mg/L or higher. The appearance of this solution was colorless and transparent.

Concentration of the test substance in the test solution

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

Observation of the test solution and microscopic observation of the test organisms

As the result of microscopic observation, no abnormalities in the cell morphology were observed from the start of exposure until the end of exposure.

Validity criteria fulfilled:
yes
Conclusions:
50% growth inhibitory concentration (ErC50) and maximum no observed effect concentration (NOECr) obtained from the growth rate at the 72-hour exposure period were ErC50 29 mg/L and NOECr 5.00 mg/L.
Executive summary:

A GLP-growth inhibition test of 2,5-xylenol in algae throughout the 72-hour exposure period in accordance with the Growth Inhibition Test of in Algae described in "Methods of Tests pertaining to New Chemical Substances, Etc" and comparable to OECD Guideline 201 has been conducted. The test has been performed with the algae Pseudokirchneriella subcapitata at an initial cell concentration of 5005 cells/mL under static conditions and for 72 hours. The concentrations of the test substance in the test solutions having 5.00, 10.0, 20.0, 40.0, and 80.0 mg/L in set concentration were 4.75, 9.62, 18.6, 38.0, and 76.3 mg/L at the start of exposure and 4.78, 9.54, 17.9, 37.6, and 74.4 mg/L 72 hours after exposure. The proportion with respect to the set concentration was in the range of 93.0-96.2% at the start of exposure and became 89.5-95.6% 72 hours after exposure, with the variation less than ± 20%. Thereby, 50% growth inhibitory concentration (ErC50) was calculated using the set concentration as the concentration of the test substance during the exposure period.

50% growth inhibitory concentration (ErC50) and maximum no observed effect concentration (NOECr) obtained from the growth rate at the 72-hour exposure period were shown below.

Exposure period

ErC50

(mg/L)

95% confidence limit (mg/L)

NOECr

(mg/L)

0-72 hours

29

26-32

5.00

Description of key information

Growth inhibition test in algae, Pseudokirchneriella subcapitata, 72h, comparable to OECD Guideline 201, GLP, static: ErC50 (72h) = 29 mg/L; NOECr (72h) = 5.00 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
29 mg/L
EC10 or NOEC for freshwater algae:
5 mg/L

Additional information

A GLP-growth inhibition test of 2,5-xylenol in algae throughout the 72-hour exposure period ( KS_Growth inhibition test in algae_Pseudokirchneriella subcapitata_72h _2008) in accordance with the Growth Inhibition Test of in Algae described in "Methods of Tests pertaining to New Chemical Substances, Etc" and comparable to OECD Guideline 201has been conducted. The test has been performed with the algae Pseudokirchneriella subcapitata at an initial cell concentration of 5005 cells/mL under static conditions and for 72 hours. The concentrations of the test substance in the test solutions having 5.00, 10.0, 20.0, 40.0, and 80.0 mg/L in set concentration were 4.75, 9.62, 18.6, 38.0, and 76.3 mg/L at the start of exposure and 4.78, 9.54, 17.9, 37.6, and 74.4 mg/L 72 hours after exposure. The proportion with respect to the set concentration was in the range of 93.0-96.2% at the start of exposure and became 89.5-95.6% 72 hours after exposure, with the variation less than ± 20%. Thereby, 50% growth inhibitory concentration (ErC50) was calculated using the set concentration as the concentration of the test substance during the exposure period.

50% growth inhibitory concentration (ErC50) and maximum no observed effect concentration (NOECr) obtained from the growth rate at the 72-hour exposure period were shown below.

Exposure period

ErC50

(mg/L)

95% confidence limit (mg/L)

NOECr

(mg/L)

0-72 hours

29

26-32

5.00