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

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:

2010-01-15 to 2010-01-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed OECD study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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-methylbenzenesulphonamide

EC Number:

256-435-3

EC Name:

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

Cas Number:

49701-24-8

Molecular formula:

C9H14N2O4S

IUPAC Name:

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

Details on test material:

- Name of test material (as cited in study report): Chinonbasesulfmethylamid
- Molecular formula: C9H14N2O4S
- Molecular weight: 246.29 g/mol
- Physical state: Solid, dark brown wet powder
- Analytical purity: 91.5 % (w/w)
- Expiration date of the lot/batch: 2010-07-29
- Storage condition of test material: Room temperature, protected from moisture and light.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The test item concentrations were analytically determined from all test concentrations and the control after 0 h (test start) and 72 h (test end).
- Sampling method: Separate samples were prepared at test start. Samples at end of the test 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* of 550 mg/L was prepared with dilution water. The dispersion was shaken with 20 rpm for 48 h at ambient temperature and filtrated with 0.45 µm RC filter.
- Eluate: Dilution water
- Differential loading: The saturated solution and further four dilution levels were tested in a geometrical series with a dilution factor √10: 1.0 - 3.2 - 10 - 32 and 100 % of the saturated solution corresponding to geometric mean measured test item concentrations of 0.304 – 0.991 – 3.45 – 11.7 – 40.2 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:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 21.5 °C, Min: 21 °C, Max: 22 °C

pH:

Geometric mean measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
40.2 7.70 7.80
11.7 7.76 7.81
3.45 7.80 7.90
0.991 7.78 7.90
0.304 7.82 7.91
Control 7.83 8.01

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods incl. tables"

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 964055 cells/mL
- No. of vessels per concentration (replicates): 3
- 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)

Dilution level of the saturated solution
[%] Rate Related Inhibition[%] Yield Inhibition [%]
100 18 61
1 3 14
0.1 1 4

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

40.2 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

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 rates and yield.
One Way Analysis of Variance and DUNNETT’s test was 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 %. These findings were marked “biologically not significant”. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % are significant. The findings were marked “biologically significant”.


EC-values and statistical EC20-values of the yield inhibition after 72 h were calculated by polynomial regression. Calculation of the confidence intervals were carried from the best fit values, the standard error and the t-distribution with the software GraphPad Prism.

Any other information on results incl. tables

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

                   based on geometric mean measured test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	40.2
LOEC	> 40.2
ErC10	> 40.2
ErC20	> 40.2
ErC50	> 40.2
	Inhibition of Yield
NOEC	0.304
LOEC	0.991
EyC10	0.304 EC10 0.991
EyC20	0.444 ( 0.304 – 0.731)
EyC50	> 40.2

Cell Densities

	Geometric mean measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
40.2		1		5074		41498	150317	616466
		2		5074		50334	199455	628069
		3		5074		42470	181210	602342
		Mean		5074		44767	176994	615626
11.7		1		5074		44512	149234	534794
		2		5074		46090	174625	549525
		3		5074		43569	194494	613321
		Mean		5074		44724	172784	565880
3.45		1		5074		50313	152850	521207
		2		5074		48345	196544	775858
		3		5074		44844	179357	624666
		Mean		5074		47834	176250	640577
0.991		1		5074		33762	184035	601235
		2		5074		46759	156175	582657
		3		5074		36681	195790	670689
		Mean		5074		39067	178667	618194
0.304		1		5074		38005	198246	908128
		2		5074		40150	177598	811220
		3		5074		30006	205667	878735
		Mean		5074		36054	193837	866028
Control		1		5074		38001	172748	929649
		2		5074		32532	136967	846095
		3		5074		34098	170337	951243
		4		5074		32081	190361	1000743
		5		5074		33159	166307	983994
		6		5074		32269	172920	1072603
		Mean		5074		33690	168273	964055

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]		[%]
40.2	1		1.60	8.48		611392	36.3
	2		1.61	8.12		622995	35.0
	3		1.59	8.92		597268	37.7
	Mean	(+)*	1.60	8.51	(+)	610552	36.3
11.7	1		1.55	11.2		529720	44.8
	2		1.56	10.7		544451	43.2
	3		1.60	8.57		608247	36.6
	Mean	(+)*	1.57	10.1	(+)	560806	41.5
3.45	1		1.54	11.7		516133	46.2
	2		1.68	4.09		770784	19.6
	3		1.60	8.22		619592	35.4
	Mean	(+)*	1.61	8.00	(+)	635503	33.7
0.991	1		1.59	8.95		596161	37.8
	2		1.58	9.55		577583	39.8
	3		1.63	6.87		665615	30.6
	Mean	(+)*	1.60	8.46	(+)	613120	36.1
0.304	1		1.73	1.09		903054	5.83
	2		1.69	3.24		806146	15.9
	3		1.72	1.72		873661	8.90
	Mean	(-)	1.71	2.02	(-)	860954	10.2
Control	1		1.74			924575
	2		1.71			841021
	3		1.75			946169
	4		1.76			995669
	5		1.76			978920
	6		1.79			1067529
	Mean		1.75			958981

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	2.01	1.51	1.68	1.74	0.254	14.6	9.16
	2	1.86	1.44	1.82	1.71	0.233	13.7
	3	1.91	1.61	1.72	1.75	0.150	8.59
	4	1.84	1.78	1.66	1.76	0.0938	5.32
	5	1.88	1.61	1.78	1.76	0.134	7.61
	6	1.85	1.68	1.83	1.79	0.0925	5.18
				Mean	1.75
				± SD	0.03
				CV [%]	1.52

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Chinonbasesulfmethylamid was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (geometric mean measured test item concentrations): The NOEC-values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 40.2 and 0.304 mg/L, respectively. The EC50-values for both inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were > 40.2 mg/L. All effect levels are given based on geometric mean measured concentrations of Chinonbasesulfmethylamid.

Executive summary:

The toxicity of Chinonbasesulfmethylamid(batch number DALA 060326) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from January 15 to 27, 2010 with the definitive exposure phase being conducted from January 18 to 21, 2010. 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 definitive test with the saturated solution and further four dilution levels were tested in a geometrical series with a dilution factor of 2, nominal: 1 - 3.2 - 10 - 32 - 100 % of the saturated solution corresponding to geometric mean measured test item concentrations of 0.304 – 0.991 – 3.45 – 11.7 – 40.2 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of Chinonbasesulfmethylamidwere analysed at all concentration levels after 0 and 72 h via HPLC analysis. The measured concentrations of Chinonbasesulfmethylamidat test end were in the range of 56 – 90 % of the initially measured concentrations. All effect values are given based on geometric mean measured concentrations of the test item.

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

                   based on geometric mean measured test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	40.2
LOEC	> 40.2
ErC10	> 40.2
ErC20	> 40.2
ErC50	> 40.2
	Inhibition of Yield
NOEC	0.304
LOEC	0.991
EyC10	0.304 EC10 0.991
EyC20	0.444 ( 0.304 – 0.731)
EyC50	> 40.2