4-anilino-3-nitro-N-phenylbenzenesulphonamide

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: FAT 36014/Z
Batch number: Q30661AASY
EAS Test item code: 2014-000181
Appearance / colour: powder / yellow
Chemical name: 3-Nitro-n-phenyl-4-(phenylamino)benzene sulfamide
CAS number: 5124-25-4
Purity analysed: 94.0 % w/w
Molecular weight: 369.40 g/mol
Date of analysis: 07 Aug 2013
Expiry date: 08 Aug 2018
Storage conditions: ambient (+5 to +30 °C), dark, dry

Analytical monitoring:

yes

Details on sampling:

The test medium samples (100 mL) were stored deep-frozen (below -18 °C) until analysis

Vehicle:

no

Details on test solutions:

Due to low water solubility of the test item a saturated solution was prepared by weighing 100 mg FAT 36014/Z TE on a weighing scoop and transferring it to a volumetric flask. Test medium was added up to the bench mark (1 L). The solution was stirred for 48 hours in the dark. Afterwards the stock solution was withdrawn from the middle of the preparation vessel by suction without transferring undissolved test item (according to OECD Sieries on Testing and Assessment No. 23). Aliquots of the withdrawn phase were diluted with test medium to receive the lower test item concentrations.

Test organisms (species):

Lemna gibba

Details on test organisms:

The test organism was the aquatic plant duckweed, Lemna gibba G3 (Alismatales: Araceae). The plants used in this study were taken as aseptic clones from the cultivation of the testing facility. They were primarily cultured by Dr. Janet Slovin, Horticulture Crops Quality Laboratory, U.S. Department of Agriculture. BARC-West, Bldg. 050 HH-4, Beltsville, MD 20705, U.S.A.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Remarks on exposure duration:

None

Post exposure observation period:

Not available

Hardness:

Not available

Test temperature:

22.9 – 25.1 °C

pH:

7.37 – 8.55

Dissolved oxygen:

Not available

Salinity:

Not available

Nominal and measured concentrations:

see table below

Details on test conditions:

Light intensity (mean): ~ 7000 lux
Light tubes: OSRAM light tubes (L 30 W77 and L 30 W25)
Exposure to light: continuously
Test vessels: 250 mL glass beakers filled with 150 mL
Number of fronds: 12 per test vessel

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.051 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield of frond number

Remarks on result:

other: 0.000217-0.183

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

0.846 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield of frond number

Remarks on result:

other: 0.270-5.80

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield of frond number

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.29 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate of frond number

Remarks on result:

other: 0.163-0.437

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate of frond number

Remarks on result:

other: -

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate of frond number

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.017 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield of dry weight

Remarks on result:

other: -

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

1.74 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield of dry weight

Remarks on result:

other: -

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.098 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield of dry weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.409 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate of dry weight

Remarks on result:

other: -

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate of dry weight

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.313 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate of dry weight

Results with reference substance (positive control):

Not available

Reported statistics and error estimates:

The statistical evaluation for day 7 was performed for yield of frond numbers, growth rate of frond numbers, growth rate dry weight and yield of dry weight. A test for normality of the data was performed by calculating the Shapiro-Wilk’s statistic, a test for homogeneity of the data was performed according to Levene. The NOEC and LOEC were determined by using a multiple comparison method (Dunnett`s t-test). The calculation of EC10 and EC20 values of all parameters was performed using probit analysis following normal and Gompertz procedure. The calculation of EC50 values for yield of frond numbers and yield of dry weight was done using probit analysis following normal procedure. For yield of dry weight the calculation was done without the lowest loading rate of 0.954 mg/L to cover only the area with a clear dose-response relation. The calculation of EC50-values for growth rate of frond numbers and dry weight were not indicated since inhibition was below 50 %. The evaluation of data was performed by SAS® (2002-2010).

Preliminary Tests (non-GLP data): Due to the low water solubility of the test item (0.03 mg/L), a 7-day static test with nominal concentrations of 100, 10 and 1 mg/L, control and solvent control was performed. Test solutions were prepared by dilution of the test item in acetone, ultrasonic treatment for 15 min and application of defined volumes of the test solutions to the test vessels.

Initial measured concentrations were found to be 63 % of nominal for the test item concentration of 1.00 mg/L and 1 % of nominal for the test item concentration of 100 mg/L. Measured concentrations in the aged test item solutions were found to be 64 % of nominal for the test item concentration of 1 mg/L and below the detection limit for the test item concentration of 100 mg/L. Based on this results the GLP-main test was performed with preparation of test solutions based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Toxicological endpoints for FAT 36014/Z:

	FAT 36014/Z [mg/L loading rate]
Endpoint	EC10	95 % confidence limit	EC20	95 % confidence limit	EC50	95 % confidence limit	NOEC^1)	LOEC^1)
Yield of frond numbers	4.23^2)	-	10.4^2)	-	59.0^2)	-	3.05	9.77
Growth rate of frond numbers	22.6^3)	0.0201-56.3	52.3^3)	-	>100	-	3.05	9.77
Yield of dry weight	9.65^2)	-	20.0^2)	-	80.3^2)	-	9.77	31.3
Growth rate of dry weight	31.1^2)	-	>100	-	>100	-	31.3	100

1) following Dunnetts-t-test

2) probit following normal procedure

3) probit following Gompertz

- not applicable

	FAT 36014/Z [mg/L loading rate]
Endpoint	EC10	EC20	EC50	NOEC	LOEC
Yield of frond numbers	4.23	10.4	59.0	3.05	9.77
Growth rate of frond no	22.6	52.3	>100	3.05	9.77
Yield of dry weight	9.65	20.0	80.3	9.77	31.3
Growth rate of dry weight	31.1	>100	>100	31.3	100
	FAT 36014/Z [mg/L] actual
Endpoint	EC10	EC20	EC50	NOEC	LOEC
Yield of frond numbers	0.0508	0.156	0.846	0.0305	0.0977
Growth rate of frond no	0.290	0.812	>2.00	0.0305	0.0977
Yield of dry weight	0.0165	0.105	1.74	0.0977	0.313
Growth rate of dry weight	0.409	>2.00	>2.00	0.313	2.00

Validity criteria fulfilled:

yes

Conclusions:

Significant inhibitory effects were determined for yield and growth rate of frond numbers at 9.77, 31.3 and 100 mg/L loading rate and 0.0977, 0.313 and 2.00 mg/L (actual), for yield of dry weight at 31.3 and 100 mg/L loading rate and 0.313 and 2.00 mg/L (actual) and for growth rate of dry weight at 100 mg/L loading rate and 2.00 mg/L (actual). The overall LOEC was based on the most sensitive parameter and was determined to be 9.77 mg/L loading rate and 0.0977 mg/L (actual). The corresponding NOEC was determined at 3.05 mg/L loading rate and 0.0305 mg/L (actual). The nominal EC10-value for yield of frond numbers is 4.23 mg/L loading rate, 9.65 mg/L loading rate for yield of dry weight, 22.6 mg/L loading rate for growth rate of frond numbers and 31.1 mg/L loading rate for growth rate of dry weight. Based on the geometric mean of the analysed concentration levels, the EC10-value for yield of frond numbers is 0.0508 mg/L, 0.0165 mg/L for yield of dry weight and 0.409 mg/L for growth rate of dry weight. The actual EC10-value for growth rate of frond numbers is 0.290 mg/L. The nominal EC20-value for yield of frond numbers is 10.4 mg/L loading rate, 20.0 mg/L loading rate for yield of dry weight, 52.3 mg/L loading rate for growth rate of frond numbers and >100 mg/L loading rate for growth rate of dry weight. Based on the geometric mean of the analysed concentration levels, the EC20-value for yield of frond numbers is 0.156 mg/L, 0.105 mg/L for yield of dry weight and > 2.00 mg/L for growth rate of dry weight. The actual EC20-value for growth rate of frond numbers is 0.812 mg/L. The nominal EC50-value for yield of frond numbers is 59.0 mg/L loading rate and 80.3 mg/L loading rate for yield of dry weight. The nominal EC50-value for growth rate of frond numbers and dry weight is >100 mg/L loading rate. Based on the geometric mean of the analysed concentration levels, the EC50-value for yield of frond numbers is 0.846 mg/L and 1.74 mg/L for yield of dry weight and > 2.00 mg/L for growth rate of dry weight. The actual EC50-value for growth rate of frond numbers and dry weight is > 2.00 mg/L. The test is valid as the doubling time of control frond numbers was calculated to be 33.2 h, corresponding to 1.385 days at day 7. This is less than the maximum allowable doubling time set in OECD guideline 221 (60 h, 2.5 days).

Executive summary:

The aim of the study was to assess the effect of the test item on the growth of Lemna gibba according to OECD Guideline 221 and determine EC10, 20, 50 and NOEC/LOEC values. A saturated stock solution was prepared by stirring 100 mg FAT 36014/Z TE in 1000 mL test medium for 48 hours in the dark. The stock solution was withdrawn from the middle of the preparation vessel. Aliquots of the withdrawn phase were diluted to receive the lower test item concentrations. A semi-static test with the following loading rates was performed: undiluted solution (corresponding to 100 mg/L loading rate), 3.2-fold diluted solution (corresponding to 31.3 mg/L loading rate), 10.2-fold diluted solution (corresponding to 9.77 mg/L loading rate), 32.8-fold diluted solution (corresponding to 3.05 mg/L loading rate), 104.9-fold diluted solution (corresponding to 0.954 mg/L loading rate) and control. Vegetative growth was observed for a test period of 7 days by measuring the increase of frond numbers and dry weight. The following endpoints (NOEC/LOEC, EC10, 20, 50) were determined:

- frond numbers and dry weight

- growth rates of frond numbers and dry weight

- yield of frond numbers and dry weight

- doubling times of frond numbers

Significant inhibitory effects were determined for yield and growth rate of frond numbers at 9.77, 31.3 and 100 mg/L loading rate and 0.0977, 0.313 and 2.00 mg/L (actual), for yield of dry weight at 31.3 and 100 mg/L loading rate and 0.313 and 2.00 mg/L (actual) and for growth rate of dry weight at 100 mg/L loading rate and 2.00 mg/L (actual). The overall LOEC was based on the most sensitive parameter and was determined to be 9.77 mg/L loading rate and 0.0977 mg/L (actual). The corresponding NOEC was determined at 3.05 mg/L loading rate and 0.0305 mg/L (actual). The nominal EC10-value for yield of frond numbers is 4.23 mg/L loading rate, 9.65 mg/L loading rate for yield of dry weight, 22.6 mg/L loading rate for growth rate of frond numbers and 31.1 mg/L loading rate for growth rate of dry weight. Based on the geometric mean of the analysed concentration levels, the EC10-value for yield of frond numbers is 0.0508 mg/L, 0.0165 mg/L for yield of dry weight and 0.409 mg/L for growth rate of dry weight. The actual EC10-value for growth rate of frond numbers is 0.290 mg/L. The nominal EC20-value for yield of frond numbers is 10.4 mg/L loading rate, 20.0 mg/L loading rate for yield of dry weight, 52.3 mg/L loading rate for growth rate of frond numbers and >100 mg/L loading rate for growth rate of dry weight. Based on the geometric mean of the analysed concentration levels, the EC20-value for yield of frond numbers is 0.156 mg/L, 0.105 mg/L for yield of dry weight and >2.00 mg/L for growth rate of dry weight. The actual EC20-value for growth rate of frond numbers is 0.812 mg/L. The nominal EC50-value for yield of frond numbers is 59.0 mg/L loading rate and 80.3 mg/L loading rate for yield of dry weight. The nominal EC50-value for growth rate of frond numbers and dry weight is >100 mg/L loading rate. Based on the geometric mean of the analysed concentration levels, the EC50-value for yield of frond numbers is 0.846 mg/L and 1.74 mg/L for yield of dry weight and > 2.00 mg/L for growth rate of dry weight. The actual EC50-value for growth rate of frond numbers and dry weight is >2.00 mg/L. The test is valid as the doubling time of control frond numbers was calculated to be 33.2 h, corresponding to 1.385 days at day 7. This is less than the maximum allowable doubling time set in OECD guideline 221 (60 h, 2.5 days).

Conclusion:

Significant inhibitory effects were determined for yield and growth rate of frond numbers at 9.77, 31.3 and 100 mg/L loading rate and 0.0977, 0.313 and 2.00 mg/L (actual), for yield of dry weight at 31.3 and 100 mg/L loading rate and 0.313 and 2.00 mg/L (actual) and for growth rate of dry weight at 100 mg/L loading rate and 2.00 mg/L (actual).

Description of key information

Significant inhibitory effects were determined for yield and growth rate of frond numbers at 9.77, 31.3 and 100 mg/L loading rate and 0.0977, 0.313 and 2.00 mg/L (actual), for yield of dry weight at 31.3 and 100 mg/L loading rate and 0.313 and 2.00 mg/L (actual) and for growth rate of dry weight at 100 mg/L loading rate and 2.00 mg/L (actual). 

Key value for chemical safety assessment

EC50 for freshwater plants:

2 mg/L

EC10 or NOEC for freshwater plants:

0.29 mg/L

Additional information

The study performed in 2014 describes toxicity of the test item 36014 to Lemna gibba was determined in a 7 day semi-static test with daily test medium renewal according to OECD TG 221. Significant inhibitory effects were determined for yield and growth rate of frond numbers at 9.77, 31.3 and 100 mg/L loading rate and 0.0977, 0.313 and 2.00 mg/L (actual), for yield of dry weight at 31.3 and 100 mg/L loading rate and 0.313 and 2.00 mg/L (actual) and for growth rate of dry weight at 100 mg/L loading rate and 2.00 mg/L (actual). The overall LOEC was based on the most sensitive parameter and was determined to be 9.77 mg/L loading rate and 0.0977 mg/L (actual). The corresponding NOEC was determined at 3.05 mg/L loading rate and 0.0305 mg/L (actual). The nominal EC10-value for yield of frond numbers is 4.23 mg/L loading rate, 9.65 mg/L loading rate for yield of dry weight, 22.6 mg/L loading rate for growth rate of frond numbers and 31.1 mg/L loading rate for growth rate of dry weight. Based on the geometric mean of the analysed concentration levels, the EC10-value for yield of frond numbers is 0.0508 mg/L, 0.0165 mg/L for yield of dry weight and 0.409 mg/L for growth rate of dry weight. The actual EC10-value for growth rate of frond numbers is 0.290 mg/L. The nominal EC20-value for yield of frond numbers is 10.4 mg/L loading rate, 20.0 mg/L loading rate for yield of dry weight, 52.3 mg/L loading rate for growth rate of frond numbers and >100 mg/L loading rate for growth rate of dry weight. Based on the geometric mean of the analysed concentration levels, the EC20-value for yield of frond numbers is 0.156 mg/L, 0.105 mg/L for yield of dry weight and > 2.00 mg/L for growth rate of dry weight. The actual EC20-value for growth rate of frond numbers is 0.812 mg/L. The nominal EC50-value for yield of frond numbers is 59.0 mg/L loading rate and 80.3 mg/L loading rate for yield of dry weight. The nominal EC50-value for growth rate of frond numbers and dry weight is >100 mg/L loading rate. Based on the geometric mean of the analysed concentration levels, the EC50-value for yield of frond numbers is 0.846 mg/L and 1.74 mg/L for yield of dry weight and > 2.00 mg/L for growth rate of dry weight. The actual EC50-value for growth rate of frond numbers and dry weight is > 2.00 mg/L. The test is valid as the doubling time of control frond numbers was calculated to be 33.2 h, corresponding to 1.385 days at day 7. This is less than the maximum allowable doubling time set in OECD guideline 221 (60 h, 2.5 days).