3-[(phenylcarbamoyl)amino]phenyl 4-methylbenzenesulfonate

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:

20 May 2019 - 23 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification and stability of the test material under storage conditions: The infrared (IR) spectrum of the test material measured at the test facility was confirmed to be identical to that provided by the sponsor. In addition, the IR spectrum of the test sample after the completion of the experiment was the same as that before the start of the experiment, and it indicated that the test material was stable under storage conditions.

The purity of the test material was treated as 100%

Analytical monitoring:

yes

Details on sampling:

- Concentration of test material in test solution: At the start of exposure, 24 and 48 hours after the start of exposure, and the end of exposure.
- Samples for measurement for concentration of test material:
1) Another solution sampled from the preparation container (at the start of exposure).
2) The test solution taken out from the test vessels in each test level for analytical chemistry (at 24 and 48 hours after the start of exposure).
3) The mixed solution taken out with equal volume of the test solution from the test vessels in each test level (at the end of exposure).
- Volume of sample: Approximately 120 mL (all test levels, at the start of exposure), 100 mL (all test levels, at 24 and 48 hours after the start of exposure), 120 mL (all test levels, at the end of exposure).

- Concentrations: The determination limit of the test material was the lowest concentration of the standard solution (0.00200 mg/L) within the range of the calibration confirmed. Therefore, the determination limit of the test material in the test solution was 0.00400 mg/L in consideration of pretreatment (dilution rate: 2).
- Sampling method: HPLC

Vehicle:

yes

Remarks:

Culture Medium

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- The test material (0.150 g) and medium (1500 mL) were mixed to produce 100 mg/L as the nominal concentration, and the mixture was stirred with a magnetic stirrer for 48 hours to prepare the test solution.
- The test solution was divided into each test vessel.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection, June 30, 1995.
- Subculture: Passage cultured under sterile conditions in this test facility.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

21.7 - 22.0 °C

pH:

7.9 - 8.1

Nominal and measured concentrations:

Nominal: 100 mg/L
Measured concentration: 0.00920 mg/L

The medium without the test material was included as a control, which was treated in the same manner as the test solution.

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flask (with gas-permeable Silicosen®)
- Fill volume: 100 mL
- Incubator: LP-0.7LEDSS (Nippon Medical & Chemical Instruments Co., Ltd., Instrument No. SIN- 009)
- Aeration: no though incubation was conducted with rotary shaking (ca 100 rpm)
- Initial cells density: 0.75 ×10^4 cells/mL
- Replicates: 6 replicates/test level (One additional test vessel for measuring the value of background was set, and one additional test vessel for analytical chemistry of the test material and was set for 24 and 48 hours, respectively.)
- Volume of test solution: 900 mL/ test level (100 mL / test vessel).

CULTURE MEDIUM
- Standard medium used: OECD medium (OECD TG 201; March 23, 2006) prepared with purified water was used.
- Detailed composition: H3BO3 (0.185 mg/L), CuCl2･2H2O (0.00001 mg/L), MnCl2･4H2O (0.415 mg/L), CaCl2･2H2O (18.0 mg/L), ZnCl2 (0.00300 mg/L), NH4Cl (15.0 mg/L), FeCl3･6H2O (0.0640 mg/L), KH2PO4 (1.60 mg/L), Na2EDTA･2H2O (0.100 mg/L), NaHCO3 (50.0 mg/L), CoCl2･6H2O (0.00150 mg/L), MgCl2･6H2O (12.0 mg/L), Na2MoO4･2H2O (0.00700 mg/L), MgSO4･7H2O (15.0 mg/L).

OTHER TEST CONDITIONS
- Light intensity and quality: Continuous illumination provided by alternative LED light to fluorescent lamp: 86 - 88 μmol·m^-2·s^-1.

EFFECT PARAMETERS MEASURED
- Cell growth: Biomass (chlorophyll fluorescence value), every 24 hours after the start of exposure (the blank correction was conducted by measuring the value of blank solution in each test level).
- Appearance of test solution: Observation at the start and end of exposure.
- pH: Another solution sampled separately from the preparation container was measured (at the start of exposure). One test vessel in each test level was measured (at the end of exposure).
- Culture temperature: It was measured at the start, 1-day and 2-day after the start, and the end of exposure.
- Light intensity: It was measured at the start, 1-day and 2-day after the start, and the end of exposure in incubator.
- Concentration of test material in test solution: At the start of exposure, 24 and 48 hours after the start of exposure, and the end of exposure.

TEST CONCENTRATIONS
- Preliminary study: The nominal concentration was 100 mg/L, the test sample and medium were mixed and stirred for 48 hours, After settlement for 1 hour, the middle layer was taken out and filtered with a membrane filter (GV, 0.22 μm pore size, Merck) by suction. The filter was treated with the test solution of about 100 mL beforehand. The solubility of the test material in medium was determined to be 0.0142 mg/L.
- Main test: Therefore, a limit test around the solubility in medium was conducted at the nominal concentration of 100 mg/L.

TREATMENT OF RESULT
- Calculation of concentration-inhibition rates:
The mean value of biomass for each test level was plotted against time to produce growth curves. Using this curve, inhibition rates were calculated comparing with control values on growth rate. The specific growth rate for a specific period was calculated as the logarithmic increase in biomass according to the following formula:

μi-j = (lnXj - lnXi) / (tj - ti)

Where:
μi-j = Specific growth rate from time i to j (normally d-1)
Xi = Value of biomass at ti : Calculated value corresponding to the initial cell concentration was
used at the start of the exposure (t0).
Xj = Value of biomass at tj
ti = Time (d) of ith measurement after beginning of exposure
tj = Time (d) of jth measurement after beginning of exposure

Specific growth rate over the exposure duration (0-72h) was calculated for determination of EC50 and NOEC. In the control, specific growth rates for section-by-section were calculated for check of validity of the test. The percentage inhibition for each exposure level was mean value of the percent inhibition in average specific growth rate for a replicate (Iμ) in test level. The percent inhibition (Iμ) was calculated from mean value for average specific growth rate in control (μc), average specific growth rate for each replicate in exposure level (μT), and the following formula:

Iμ = (μc -μT) / μc X 100

- Estimation of EC50:
The EC50 was estimated as "> the test concentration (solubility in medium)" since no less than 50 % of inhibition rate was not obtained within the exposure level. The EC50 was denoted as ErC50 based on growth rate. The results of this study were estimated based on the time weighted mean of the measured concentrations as the test concentration.

- Estimation of NOEC:
Regarding the growth rate, after F test was done to determine the homogeneity of variance for the data, Aspin-Welch t-test was used to estimate the significant difference in comparison with the control. The statistical analysis was conducted using computer program (running on Microsoft software “Excel”) constructed by the test facility. The NOEC was determined by the results of statistical analysis and cell condition. The NOEC was estimated as “> the test concentration (solubility in medium)” since the growth inhabitation was not observed in the exposure level.

VALIDITY OF TEST
- The test was considered valid if the following criteria were met:
a) The cell growth in the control cultures should have increased by a factor of at least 16 within the 72-hour exposure period.
b) The mean coefficient of variation for section-by-section specific growth rates in the control cultures must not exceed 35 % during 72-hour exposure.
c) The coefficient of variation of average specific growth rates in replicate control cultures must not exceed 7 % at 72 hours after the start of exposure.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.009 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.009 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

OBSERVATION AND MEASUREMENT OF TEST SOLUTION
- Appearance: At the start of exposure, test solutions of exposure level was white suspension with suspended matter. At the end of exposure, the appearance of test solution in exposure level was green with due to the algal growth containing white suspension. The solution of control was colourless and clear at the start of exposure, and it was green at the end of exposure due to the algal growth.
- Concentration of test material in test solution: The measured concentrations of test material in the test solutions were 0.0146 mg/L at the start of exposure, 0.00543 mg/L at 24 hours after the start of exposure, 0.00830 mg/L at 48 hours after the start of exposure, and 0.0156 mg/L at the end of exposure. Those were 37.1 %, 56. 8% and 107 % at the start of exposure, respectively. The time weighted mean of the measured concentration was 0.00920 mg/L.

GROWTH CURVES AND NOEC
- The algal growth in exposure level was same as the control. The following results of cell observation were based on the comparison with the control. The conditions of cells in all exposure levels were same as the control. In the control, the condition of cells was not abnormal. On the growth rate, there was no statistical difference in exposure level. By the results in statistical analysis and cell observation showed above, NOEC based on the growth rate was estimated at ≥ 0.00920 mg/L.

VALIDITY OF THE TEST
- Growth of control The cell in the control grew exponentially during the exposure. At the end of exposure, it increased to a factor of 128 or more of the number of initial cells in the control. This meets the validity of test: the cell growth in the control should have increased by a factor of at least 16 at 72 hours after the start of exposure.
- Specific growth rates of section-by-section in controls: The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 8.3 %. It meets the validity of test: the mean coefficient of variation in the control cultures must not exceed 35 %.
- Specific growth rates in replicate controls: The coefficient of variation of specific growth rates in replicate control cultures was 2.2 %. It meets the validity of test: the coefficient of variation in control cultures must not exceed 7 %.

Results with reference substance (positive control):

The positive control study was conducted from 26 to 29 March 2019. The 72-hour ErC50 was determined to be 1.1 mg/L. This value was within the stipulated range (mean ± 2 S.D.: 0.61 - 1.4 mg/L, n=32, fluorescent lights) to background data at the Test Facility.

Reported statistics and error estimates:

EC50 (ErC50): > 0.00920 mg/L
NOEC: ≥ 0.00920 mg/L

Value of biomass at each time

Measured concentration (mg/L)	No.	Chlorophyll fluorescence value (relative unit)
		0 hour^b	24 hours	48 hours	72 hours
Control	A B C D E F Mean S.D.	46 46 46 46 46 46 46 0	270 280 270 270 270 270 270 2.8	1500 1600 1600 1700 1600 1600 1600 60	5900^c 7900 7700 8000 7300 7800 7500 790
0.00920	A B C D E F Mean S.D.	46 46 46 46 46 46 46 0	270 270 260 270 280 280 270 5.4	1600 1600 1600 1600 1600 1500 1600 25	7100 7500 8200 7800 7800 7300 7600 380

b： The value based on the measured value of pre-culture

c： The minimum cell growth in the control (biomass at the end of exposure / biomass at the start of exposure) 5900 / 46 = 128

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 72-hour EC50 of the test material (based on growth rate) was determined to be > 0.00920 mg/L based on the time weighted mean of measured concentration. The 72-hour NOEC (based on growth rate) was ≥ 0.00920 mg/L.

Executive summary:

The effect of the test material on the test organisms, Pseudokirchneriella subcapitata, was investigated in accordance with the standardised guidelines OECD 201, and under GLP conditions. 

This study was conducted as a limit test in order to estimate the effect on the test organisms under the solubility of test material in medium. As a result, no effect was found in the definitive study. Therefore, it was decided that the test material had no remarkable effect on the test organisms at around the solubility in medium. Since the test material concentration in the test solutions were expected to decrease during the exposure by the preliminary test results, the test material concentration in the test solutions were measured at the start of the exposure, 24 and 48 hours after the start of exposure, and the end of exposure. As a result, the test material concentration tended to decrease, but the test material concentration in the test solution was nearly the solubility in the medium during the exposure. Also, the increase in the concentration of the test material in the test solution observed after 48 hours of exposure were considered to be because the test material present as an insoluble matter in the test solution was dissolved under the test conditions. The environmental conditions were within the suitable range. Therefore, it was concluded that this study complied with the applied test guidelines.

Under the conditions of this study, the 72-hour EC50 of the test material (based on growth rate) was determined to be > 0.00920 mg/L based on the time weighted mean of measured concentration. The 72-hour NOEC (based on growth rate) was ≥ 0.00920 mg/L.

Description of key information

The 72-hour EC50 of the test material (based on growth rate) was determined to be > 0.00920 mg/L based on the time weighted mean of measured concentration. The 72-hour NOEC (based on growth rate) was ≥ 0.00920 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.009 mg/L

Additional information

The effect of the test material on the test organisms, Pseudokirchneriella subcapitata, was investigated in accordance with the standardised guidelines OECD 201, and under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

This study was conducted as a limit test in order to estimate the effect on the test organisms under the solubility of test material in medium. As a result, no effect was found in the definitive study. Therefore, it was decided that the test material had no remarkable effect on the test organisms at around the solubility in medium. Since the test material concentration in the test solutions were expected to decrease during the exposure by the preliminary test results, the test material concentration in the test solutions were measured at the start of the exposure, 24 and 48 hours after the start of exposure, and the end of exposure. As a result, the test material concentration tended to decrease, but the test material concentration in the test solution was nearly the solubility in the medium during the exposure. Also, the increase in the concentration of the test material in the test solution observed after 48 hours of exposure were considered to be because the test material present as an insoluble matter in the test solution was dissolved under the test conditions. The environmental conditions were within the suitable range. Therefore, it was concluded that this study complied with the applied test guidelines.

Under the conditions of this study, the 72-hour EC50 of the test material (based on growth rate) was determined to be > 0.00920 mg/L based on the time weighted mean of measured concentration. The 72-hour NOEC (based on growth rate) was ≥ 0.00920 mg/L.