4-amino-2,5-dimethoxy-N-phenylbenzenesulphonamide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-09-25 to 2009-10-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline conform study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-2,5-dimethoxy-N-phenylbenzenesulphonamide

EC Number:

257-821-4

EC Name:

4-amino-2,5-dimethoxy-N-phenylbenzenesulphonamide

Cas Number:

52298-44-9

Molecular formula:

C14H16N2O4S

IUPAC Name:

4-amino-2,5-dimethoxy-N-phenylbenzene-1-sulfonamide

Details on test material:

- Name of test material (as cited in study report): Chinonbasesulfanilid, trocken
- Molecular formula: C14H16N2O4S
- Molecular weight : 308.36 g/mol
- Physical state: Solid, grey powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The limit concentration and the control were analytically verified at the beginning and the end of the test.
- Sampling method: Samples at test start and end were taken directly from the test replicates.
- Sample storage conditions before analysis:All samples were stored at 6 ± 2 °C until start of analysis, if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution was prepared. A dispersion of 360 mg/L prepared in dilution water was shaken with 20 rpm for 48 h at ambient temperature and filtrated with 0.45 µm RC filter.
- Eluate: Dilution water
- Differential loading: Limit concentration, geom. mean measured: 8.46 mg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 22.5 °C, Min: 22 °C, Max: 22 °C

pH:

Geometric mean measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
8.46 8.18 8.55
Control 8.07 8.56

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Concentrations and Recovery Rates of Chinonbasesulfanilid, trocken

Sampling date 2009-09-28
0 h 2009-10-01
72 h
Start of analysis 2009-09-28 2009-10-01
Chinonbasesulfanilid, trocken
Geometric mean
measured conc.
[mg/L]
Measured conc.
[mg/L] Measured conc.
[mg/L] RR
[%]
Saturated Solution 18.1 3.95 22 8.46
Control < LOQ < LOQ —

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 5074 cells/mL
- Control end cells density: Mean 1184583 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes

Results of the Range Finding Test (0 – 72 h)

[%] Saturated Solution Rate-related Inhibition
[%] Yield Inhibition

[%]
100 -2 -10
10 1 4
1 1 5

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8.46 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: Yes

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.600 (CI 0.545 - 0.653) mg/L
Yield Inhibition: 0.280 (CI 0.266 - 0.299) mg/L

Reported statistics and error estimates:

The NOEC and LOEC were determined by calculation of statistical significance of growth rate and yield. One Way Analysis of Variance and DUNNETT’S test were carried out for the determination of statistically significant differences compared to control replicates. When running a One Way Analysis of Variance a Normality Test and an Equal Variance Test were done first. P-values for both Normality and Equal Variance Tests were 0.05. The a-value for ANOVA was a=0.05. Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 %. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % were marked biological significant.

Any other information on results incl. tables

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Chinonbasesulfanilid, trocken
(0-72 h)

                   based on the geometric mean measured test item concentration [mg/L] of the saturated solution

                               

	Rate-related Inhibition
NOEC	8.46
LOEC	> 8.46
ErC10	> 8.46
ErC20	> 8.46
ErC50	> 8.46
	Inhibition of Yield
NOEC	8.46
LOEC	> 8.46
EyC10	> 8.46
EyC20	> 8.46
EyC50	> 8.46

Cell Densities

	Geometric mean measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
8.46		1		5074		42097	305374	1362580
		2		5074		39289	243116	1327988
		3		5074		45984	262087	1132426
		4		5074		39186	255256	1393941
		5		5074		40428	291475	1350387
		6		5074		41937	325690	1469783
		Mean		5074		41487	280500	1339518
Control		1		5074		43479	294526	1381862
		2		5074		38571	302849	1134259
		3		5074		35976	221624	1125042
		4		5074		43471	240570	1072517
		5		5074		39473	217700	1147195
		6		5074		43639	295731	1246620
		Mean		5074		40768	262167	1184583

Evaluation after 72 h

              Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

                              

Geometric mean measured test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
8.46	1		1.86	-2.63		1357506	-15.1
	2		1.86	-2.16		1322914	-12.2
	3		1.80	0.76		1127352	4.42
	4		1.87	-3.05		1388867	-17.8
	5		1.86	-2.47		1345313	-14.1
	6		1.89	-4.02		1464709	-24.2
	Mean	(+)*	1.86	-2.26	(+)*	1334444	-13.1
Control	1		1.87			1376788
	2		1.80			1129185
	3		1.80			1119968
	4		1.79			1067443
	5		1.81			1142121
	6		1.84			1241546
	Mean		1.82			1179509

Section-by-Section and Average Specific Growth Rates of the Control Group
               (0 – 72 h)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 h	24 - 48 h	48 - 72 h
Control	1	2.15	1.91	1.55	1.87	0.304	16.2	16.5
	2	2.03	2.06	1.32	1.80	0.418	23.2
	3	1.96	1.82	1.63	1.80	0.168	9.32
	4	2.15	1.71	1.50	1.79	0.333	18.6
	5	2.05	1.71	1.66	1.81	0.213	11.8
	6	2.15	1.91	1.44	1.84	0.363	19.8
				Mean	1.82
				SD ±	0.03
				CV [%]	1.68

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Chinonbasesulfanilid, trocken was found not to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus after 72 h tested with the saturated solution at 8.46 mg/L. For details of effect values please refer to Table 1. All effect levels are given based on geometric mean measured concentrations of Chinonbasesulfanilid, trocken.

Executive summary:

The toxicity of Chinonbasesulfanilid, trocken to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

The study was conducted under static conditions with an initial cell density of approximately  2 – 5 x 10³cells/mL. Based on a preliminary test, a limit test with the saturated solution was carried out. Six replicates were tested for limit concentration and control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of Chinonbasesulfanilid, trocken were analysed at the limit concentration level (the saturated solution) at test start and test end via HPLC-DAD analysis. The measured concentrations of the test item at test start and end were 18.1 and 3.95 mg/L, respectively. All effect values are given based on the geometric mean measured concentration of the test item. Chinonbasesulfanilid, trocken caused a stimulation of growth.

Table 1:    NOEC, LOEC, EC - values and 95 % Confidence Intervals of Chinonbasesulfanilid, trocken
(0-72 h)

                   based on the geometric mean measured test item concentration [mg/L] of the saturated solution

                               

	Rate-related Inhibition
NOEC	8.46
LOEC	> 8.46
ErC10	> 8.46
ErC20	> 8.46
ErC50	> 8.46
	Inhibition of Yield
NOEC	8.46
LOEC	> 8.46
EyC10	> 8.46
EyC20	> 8.46
EyC50	> 8.46