Rabitle FP-100

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date: 20th July 2004 Experimental start date: 20th July 2004 Experimental end date: 23rd July 2004 Study completion date: 1st October 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: - Study generated according to generally valid and/or internationally accepted testing guidelines - Performed according to GLP - Test parameters based on specific testing guideline - No GLP certificate provided

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

Algal Growth Inhibition Test stipulated in the ‘Testing Methods for New Chemical Substances’ (November 21, 2003; No. 1121002. Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; November 13, 2003,

Deviations:

no

Principles of method if other than guideline:

This study was performed in compliance with:

"Standard Concerning Testing Facility Relting to New
Chemical Substances" (November 21, 2003; No. 1121003,
Pharmaceutical and Food Safety Bureau, Ministry of Health,
Labour and Welfare: November 17, 2003, No.3, Manufacturing
indutries Bureau, Ministry of Economy, Trade and Industry;
No. 031121004, Environmental Policy Bureau, Ministry of the
Environment).

OECD Principles of Good Laboratory Practice (November 26,
1977)


OECD guideline 201 Freshwater Alga and Cyanobacteria, Growth
Inhibition Test

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
N/A

Analytical monitoring:

yes

Details on sampling:

See Reprt

Vehicle:

yes

Details on test solutions:

The medium mentioned in ‘Method for Alga, Growth Inhibition Test of Chemical Substances” stipulated in the “Testing Methods for New Chemical Substances” November 21, 2003; No. 1121002, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; November 13, 2003, No. 2,
Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 031121002, Environmental Policy Bureau, Ministry of the Environment was used for the per-culture and the test.

Medium with sterile condition was used.

Reason for setting test concentration:
In the preliminary measurement of solubility to medium, the solubility examined showed large variation and it was not able to determine a constant solubility. In the preparation method of test solution by stirring, a 48-hour period, it was difficult to prepare a suitable test solution because the saturated concentration of the test item varied greatly (0,300 to 0.0300 mg/I.). The 1.00 and 0.100 mg/I. test solutions which were prepared using N,N-Dimethylformamide (DMF) were maintained each nominal concentration at preparation, and these concentrations were maintained the maximum solubility (0.300 mg/L) and the minimum solubility (0.0300 mg/I.). The nominal concentration of 1.00, 0.316 and 0.100 mg/I. prepared with DMF was used in this test in consideration for the results of preliminary tests (see Additional data).

Preparation of test solution:
Correction with the purity (99.3%) was not applied to the preparation of test solution. Desired amount of the test item weighed was dissolved in DMF to prepare a primary stock solution of 10,000 mg/L, This stock solution was diluted with DMF, and secondary stock solutions of 3,160 and 1,000 mg/L were prepared. A required quantity of the stock solution was taken and mixed with medium in container for preparation, and both were agitated. The prepared solutions were divided into each test vessel.

Volume of test solution:
300 mL/exposure level and control (3 replicates of 100 mL/vessel)
600 mL/vehicle control (6 replicates of 100 mL/vessel)

Identity and concentration of auxiliary solvent for dispersal: N,N-Dimethylformamide (DMF)

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): Pseudokirchnerella subcapitata which originally came from the American Type Culture Collection (12301 Parklawn Drive Rockville, Maryland 20852-1776 USA) on June 30th 1995 and have been cultured in Kurume Laboratory were used.
- Age of inoculum (at test initiation): No details provided in report
- Method of cultivation: An algal growth inhibition test of K2Cr2O7 (Reagent grade, Wako Pure Chemical Industries, Ltd.) with the test organism was conducted to confirm the reproducibility of the test system. The EbC50 (0-72h) and ErC50 (0-3d) of K2Cr2O7 were 0.376 and >0.800 mg/L respectively and EbC50 (0-72h): 0.376 mg/L was within the stipulated range of normal data that have been obtained in this laboratory.

ACCLIMATION
- Acclimation period: No details provided in report
- Any deformed or abnormal cells observed: No details provided in report

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

N/A

Hardness:

No details provided

Test temperature:

22.8 – 23.3°C in incubator

pH:

8.3 - 8.4 at start of exposure and 7.8-7.9 at end of exposure

Dissolved oxygen:

No details provided

Salinity:

N/A

Nominal and measured concentrations:

Three exposure levels: 1.00, 0.316 and 0.100 mg/L ( a geometric series with a factor of √10)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterilised 500 mL Erlenmeyer flask with airtight plug
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume:
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: The pre-culture, incubated under the same conditions as the test for 3 days and exponentially growing was used as inoculum to prepare the initial cell concentration of 104 cells/mL.
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
The medium mentioned in ‘Method for Alga, Growth Inhibition Test of Chemical Substances” stipulated in the “Testing Methods for New Chemical Substances” (November 21, 2003; No. 1121002, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; November 13, 2003, No. 2,
Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 031121002, Environmental Policy Bureau, Ministry of the Environment was used for the per-culture and the test.
Medium with sterile condition was used.



TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: pH and water temperature of test solution were measured at start and end of exposure. At start of exposure, another solution prepared separately was used for the measurements. At end of exposure, measurement was carried out for one of three vessels in each exposure level. Culture temperature and light intensity in the incubator were measured once a day during the exposure. Condition of test solutions was observed at start and end of exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Light intensity and quality: 102 - 116 µE/m2s (Continuous illumination provided with 60 - 120 µE/m2s* at the level of the test solutions, not varied more than 20% using a fluorescent light with wavelength range of 400-700 nm (*120 µE/ m2s = 0.72 x1020 photons/m2s))

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): EbC50, ErC50 and NOEC

- Determination of cell concentrations [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: Cell concentration was counted with particle counter at 24, 48, and 72 hours after the start of the exposure. In order to measure the blank value of the test solution in each exposure level, it was measured simultaneously about the test vessel (with no Algae) separately prepared to blank value measurement, and used for compensation. Furthermore, the cell condition for one vessel in each exposure level was observed under microscope at the end of the exposure.
- Chlorophyll measurement: No details provided in report

TEST CONCENTRATIONS

- Results used to determine the conditions for the definitive study:

1. Solubility to test medium: Test medium including an excessive amount of the test item (direct addition equivalent of 100 mg/I) was stirred for 24 and 48 hours under 23±1°C. After stirring, each solution was settled at room temperature. The water samples from middle of the aqueous layer at 24 and 48 hours were filtered by 0-140 and centrifuged (10,000xg, 30 mm) to remove the insoluble materials for analysis. The test item was detected as the main peak (81.4 wt%) of four components on the chromatogram under the analytical conditions.

The solubility to medium measured showed large variation due to low solubility of the test item and it was not able to determine a constant solubility. Therefore, the measurement of solubility to medium is not conducted.

2. Examination of preparation of test solution and effect on test organisms: i) Preparation of saturated solution
In the preparation method of ultrasonic irradiation and stirring, a 48-hour period, it was difficult to prepare a suitable saturated solution because the saturated concentration of the test item varied greatly. ii) Preparation of test solution using DMF
After stock solutions of 10,000, 3,000 and 1,000 mg/I. which were prepared using DMF were prepared, the stock solutions were diluted with medium to produce the test solutions of 1.00, 0.300 and 0.100 mg/I. and stability of these solutions were confirmed. Also effect on the test organisms was investigated in the same solution. Pre-treatment of these test solutions for analysis were conducted by fi1trations with a 0-140 and centrifugation (10,000xg, 30 min) to remove the insoluble materials.

Results can be found in Additional data in attached report.

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.643 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.643 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.643 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.643 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Meas. (TWA) - The results of the test were estimated on the basis of time-weighted mean of the measured concentration because each measured concentration during the exposure was not kept within ±20 per cent of the nominal concentration. The vehicle control was used in the calculation of inhibition rate as control.
After Bartlett’s test was done to determine the homogeneity of variance for the data based on area under growth curve and growth rate, one-way ANOVA was used to estimate the significant difference in comparison with the control. NOEC was determined in consideration of the whole test results in addition to these results of statistical analysis.

Reported statistics and error estimates:

Preparation of calibration curve:
The standard solutions of 0.0100, 0.0500, 0.100, and 0.200 mg/L were prepared by the same procedure as described for standard solution. These standard samples were analyzed according to the quantitative analytical conditions described for method of analysis. A calibration curve was drawn from the relationship between the test item concentration and the peak area on the chromatogram, respectively. The determination limit of the test item was 0.0100 mg/L based on the lowest determination-limit of the standard solutions within the range of the calibration confirmed. Therefore, the lowest determination-limit of the test item in the test solution was 0.0200 mg/L in consideration of the pre-treatment method.

Values were rounded off in accordance with JIS Z 8401 rule B.
(JIS ; Japanese Industrial Standards)

See Report

Validity criteria fulfilled:

yes

Conclusions:

Growth of the control: The cell in the control grew exponentially during the exposure. At the end of exposure, it increased to 144 to 145 or more times of the number of initial cells in the control and the vehicle control. This meets the validity of the test: the cell growth in control should have increased by a factor of at least 16 within the 72-hour exposure period.

The specific growth rate for section - by - section in the controls: The specific growth rates of section - by - section in the control and vehicle control were 1.64 and 1.67 (0-1d), 1.73 and 1.73 (1-2d) and 1.72 and 1.70 (2-3d), respectively. The mean and the standard deviation of all specific growth rates in the control and vehicle control were 1.70±0.05 and 1.70±0.03, respectively. The mean coefficients of variation in the control and vehicle control were 3.01 and 1.76%, respectively. They meet the validity of the test: the mean coefficient of variation in the control must not exceed 35%.

The specific growth rates in replicate controls: The mean and their standard deviation of specific growth rates section - by -. section in replicate in the control and vehicle control were 1.64±0.08 and 1.67±0.04 (0-1d), 1.74±0.08 and 1.73±0.03 (1-2d) and 1.73±0.03 and 1,70±0.07, respectively. The coefficients of variations were 5.11, 4.60 and 4.92% in the control, and 2.15, 1.96 and 4.34% in the vehicle control. They meet the validity of the test: the mean coefficient of variation in controls must not exceed 15%.

The present study was conducted in order to confirm the effect on the test organisms near the solubility of the test item. The measured concentrations of 0.402 mg/L in 1.00 mg/L of the highest concentration at the end of the exposure although each measured concentration during the exposure was not kept within ±20 per cent of the nominal concentration, This concentration was estimated to be higher than the expected solubility to medium. Therefore, it is concluded that there is no potential for adverse effect of the test item on the test organisms near the solubility to the medium.

Executive summary:

Algal growth inhibition test of FP-100 withPseudcikirchneriella subcapitatawas conducted. The test was performed under the following conditions: three concentrations of test item at 1.00, 0.316 and 0.100 mg/L, and a vehicle control and a control, temperature at 21-24°C (deviation of ±2°C), continuous fluorescent lamp illumination of 60-120 µE/m2s (deviation of ±20%) at surface of the test solutions. The airtight test vessels were used and continuously shaken with gyration at about 100 rpm. Algal growth was determined by the measurement of cell concentration.   The measured concentrations of the test item in the test solution at the start and end of the exposure were 96.6 to 109% and 26.0 to 40.2% of the nominal concentration, respectively. The time-weighted mean values obtained at 1.00, 0.316 and 0.100 mg/L were 0.643, 0.215 and 0.0727 mg/L, respectively, and test results were evaluated based on these values.   EbC50 (0-72h) and ErC50 (0-3d) of FP-100 calculated from the data on the area under the growth curve and growth rate were both >0.643 mg/L. No observed effect concentration (NOEC) based on each parameter was also 0.643 mg/L.   The measured concentrations was 0.402 mg/L in 1.00 mg/L of the highest concentration at the end of the exposure although each measured concentration during the exposure was not kept within ±20 per cent of the nominal concentration. This concentration was estimated to be higher than the expected solubility to test medium. Therefore, It is concluded that there is no potential for adverse effect of the test item on the test organisms near the solubility to the test medium.

Description of key information

Algal growth inhibition test of FP-100 with Pseudcikirchneriella subcapitata was conducted. The time-weighted mean values obtained at 1.00, 0.316 and 0.100 mg/L were 0.643, 0.215 and 0.0727 mg/L, respectively, and test results were evaluated based on these values.
EbC50 (0-72h) and ErC50 (0-3d) of FP-100 calculated from the data on the area under the growth curve and growth rate were both >0.643 mg/L. No observed effect concentration (NOEC) based on each parameter was also 0.643 mg/L.
The measured concentrations was 0.402 mg/L in 1.00 mg/L of the highest concentration at the end of the exposure although each measured concentration during the exposure was not kept within ±20 per cent of the nominal concentration. This concentration was estimated to be higher than the expected solubility to test medium. Therefore, It is concluded that there is no potential for adverse effect of the test item on the test organisms near the solubility to the test medium.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.643 mg/L

EC10 or NOEC for freshwater algae:

0.643 mg/L

Additional information

Algal growth inhibition test of FP-100 withPseudcikirchneriella subcapitatawas conducted. The test was performed under the following conditions: three concentrations of test item at 1.00, 0.316 and 0.100 mg/L, and a vehicle control and a control, temperature at 21-24°C (deviation of ±2°C), continuous fluorescent lamp illumination of 60-120 µE/m2s (deviation of ±20%) at surface of the test solutions. The airtight test vessels were used and continuously shaken with gyration at about 100 rpm. Algal growth was determined by the measurement of cell concentration.   The measured concentrations of the test item in the test solution at the start and end of the exposure were 96.6 to 109% and 26.0 to 40.2% of the nominal concentration, respectively. The time-weighted mean values obtained at 1.00, 0.316 and 0.100 mg/L were 0.643, 0.215 and 0.0727 mg/L, respectively, and test results were evaluated based on these values.   EbC50 (0-72h) and ErC50 (0-3d) of FP-100 calculated from the data on the area under the growth curve and growth rate were both >0.643 mg/L. No observed effect concentration (NOEC) based on each parameter was also 0.643 mg/L.   The measured concentrations was 0.402 mg/L in 1.00 mg/L of the highest concentration at the end of the exposure although each measured concentration during the exposure was not kept within ±20 per cent of the nominal concentration. This concentration was estimated to be higher than the expected solubility to test medium. Therefore, It is concluded that there is no potential for adverse effect of the test item on the test organisms near the solubility to the test medium.