3,3'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[N-cyclohexyl-2,4,6-trimethylbenzenesulphonamide]

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-09-29 to 2015-10-08

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:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3,3'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[N-cyclohexyl-2,4,6-trimethylbenzenesulphonamide]

EC Number:

245-728-1

EC Name:

3,3'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[N-cyclohexyl-2,4,6-trimethylbenzenesulphonamide]

Cas Number:

23552-74-1

Molecular formula:

C44H52N4O6S2

IUPAC Name:

3,3'-[(9,10-dioxo-9,10-dihydroanthracene-1,4-diyl)diimino]bis(N-cyclohexyl-2,4,6-trimethylbenzenesulfonamide)

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the saturated solution
The saturated solution (100 mg/L test item were weighed out) was prepared with dilution water one day prior to the start of the exposure.

Treatment
The test item was dispersed in the dilution water by stirring with approximately 1100 rpm for 24 hours at room temperature. After completion of stirring, the test item dispersion was filtered with a membrane filter (0.45 µm, RC, MACHEREY-NAGEL) to achieve the saturated solution.

Test concentration
One limit concentration was tested. The limit concentration is based on the results of a preliminary range finding test.

Control
Six replicates (without test item) were tested under the same test conditions as the test vessels.

Test organisms

Test organisms (species):

Lemna gibba

Details on test organisms:

Test organism
Duckweed, Lemna gibba G3, Lemnaceae, Arales, Arecidae, Monocotyledonae
Young, rapidly growing plants without visible lesions or discoloration (chlorosis) were used for the test.

Reason for the selection of the test organism
According to the guideline Lemna gibba is a suitable species because it is a species representative of temperate areas and is commonly used for toxicity tests.

Origin
EUROFINS-GAB GMBH, Eutinger Str. 24, 75223 Niefern-Öschelbronn, Germany.

Date of receipt 2014-08-15

Cultivation at test facility
The species was bred in the test facility. Density was kept low to prevent conglomerates of plants on the surface. At least once per week, plants were transferred to freshly prepared growth medium. Growth media and breeding vessels were autoclaved before use to enable the breeding of axenic cultures.

Breeding vessels
Crystallisation dishes of glass, vol. 900 mL, filled with ca. 500 mL growth medium, covered with glass tops.

Medium
20X-AAP-medium (Algal Assay Procedure medium), pH-value 7.5 ± 0.1

Temperature 24 ± 2 °C

Light regime Continuous fluorescent light, 15 - 60 µE•m-2•s-1

Acclimatization of the test system
The test system was held for 7 days under test conditions to acclimatize. These acclimatize plants (test system) were used in the test.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Test conditions

Test temperature:

min: 22.6
max: 23.7
mean value: 23.2

pH:

Nominal loading
of the test item
[mg/L] 0 h(Fresh medium) 2 d (Old medium) 2 d (Fresh medium) 5 d (Old medium) 5 d (Fresh medium) 7 d (Old medium)
100 7.78 8.17 7.80 8.18 7.81 8.21
Control 7.53 8.15 7.69 8.19 7.76 8.20

Nominal and measured concentrations:

Nominal loading of the test item: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: no
- Test vessel: Crystallisation dishes
- Type (delete if not applicable): covered with glass tops
- Material, size, headspace, fill volume: glass, 500 mL, with headspace, filled with 200 mL
- Type of cover: glass tops
- Aeration: no
- Agitation: no
- Renewal rate of test solution (frequency/flow rate): Semi-static, with renewal of the test solutions every 2 – 3 days (i.e. exposure to freshly prepared test and control solutions on at least two occasions during the exposure)
- Control end cells density: 192 fronds
- No. of colonies per vessel: 3
- No. of fronds per colony: 4
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h cool daylight
- Light intensity and quality: Continuous, fluorescent light, 6500 to 10000 lux (corresponding to 85 to 135 µE*m-2*s-1) on the surface of the test medium. The difference of light intensity at any measured incubation place was less than 15 % from the mean value. Actual: min: 87.2 max: 114 mean value: 102

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: manual counting
- Determination of biomass: dry weight


RANGE-FINDING STUDY
The results of the preliminary test (static) are presented below:

Table 3: Results of the Range Finding Test after 7 days
(mean values)
Nominal Test Item Concentration [%] Growth Rate Inhibition [%] Yield Inhibition [%]
10.0 -1 -1
100 -1 -3
Negative values = growth stimulation

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 7

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield frond number

Effect concentrations 8

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield frond number

Effect concentrations 9

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield frond number

Effect concentrations 10

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

other: yield dry weight

Effect concentrations 11

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield dry weight

Effect concentrations 12

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield dry weight

Details on results:

- Any visual signs of phytotoxicity (abnormalities): No
- Decrease in frond size: No
- Necrosis / chlorosis: No
- Sinking of fronds: No
- Any stimulation of growth found in any treatment: see results

Results with reference substance (positive control):

The acute toxicity of 3,5-Dichlorophenol (FLUKA, batch number SZBD168XV, purity 99.4%, CAS RN 591-35-5) to the monocotyledon Lemna gibba was determined over a period of 7 days from
2015-09-30 to 2015-10-07 according to OECD Guideline 221.

EC50-Values of the Reference Item based on the nominal concentrations [mg/L], (0-7 days)


Growth rate inhibition (number of fronds) Valid Range (average ± 3 x SD)
ErC50 5.25 5.39 ± 2.70 95% confidence interval 5.14 – 5.36
Yield inhibition (number of fronds)
EyC50 4.17 4.46 ± 2.95 95% confidence interval 2.52 – 7.22
Growth rate inhibition (dry weight)
ErdwC50 4.29 5.21 ± 2.24 95% confidence interval 2.61 – 7.24
Yield inhibition (dry weight)
EydwC50 4.08 4.44 ± 2.28 95% confidence interval 2.40 – 7.23

SD = standard deviation

Reported statistics and error estimates:

EC-values and statistical analyses
EC-values were estimated empirically based on the results of the single treatment level (limit test design).

NOEC and LOEC values
NOEC/LOEC was determined by calculation of statistical significance of inhibition of growth rates and yield in comparison to the control using: One Way Analysis of Variance (ANOVA) and DUNNETT’s test as a standard. A normality test and an equal variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The SPEARMAN rank test was used for equal variance test. P-values for both Normality and equal variance test are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

Software
All data will be computer generated and rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data, minor variations may occur from these figures.
Calculations and graphics will be carried out using the following software
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Any other information on results incl. tables

Frond Numbers

Nominal loading of thetest item [mg/L]	Repl. number	Frond number
		0 days*	2 days	5 days	7 days
100	1	12	22	69	200
	2	12	25	87	234
	3	12	27	89	222
	4	12	24	77	211
	5	12	23	71	175
	6	12	26	85	205
	Mean	12	25	80	208
Control	1	12	22	66	179
	2	12	21	67	165
	3	12	26	82	206
	4	12	23	78	189
	5	12	25	80	212
	6	12	23	75	201
	Mean	12	23	75	192

 

* = 3 colonies with 4 fronds each per replicate were inoculated at start of the exposure

 

Evaluation of Frond Numbers after 7 Days

 Statistically significant differences of growth rates and yield compared to control values are marked (+) and not significant differences are marked (-).

                                               

Nominal loading of the test item [mg/L]		Repl. No.		Growth rate [d^-1]			Inhibition of Growth rate [%]		Yield [fronds]			Inhibition of yield [%]		Doubling time [d]
100		1				0.402		-1				188		-4		1.72
		2				0.424		-7				222		-23		1.63
		3				0.417		-5				210		-17		1.66
		1				0.410		-3				199		-11		1.69
		2				0.383		3				163		9		1.81
		3				0.405		-2				193		-7		1.71
		Mean		(-)		0.407		-3		(-)		196		-9		1.71
Control		1				0.386						167				1.80
		2				0.374						153				1.85
		3				0.406						194				1.71
		4				0.394						177				1.76
		5				0.410						200				1.69
		6				0.403						189				1.72
		Mean				0.396						180				1.75

Repl. No. = Replicate number

 

 

Evaluation of Specific Growth Rate and Yield Dry Weight after 7 Days

Statistically significant differences of specific growth rates and yield compared to control values are marked (+) and not significant differences are marked (-).

 

Nominal loading of thetest item [mg/L]	Repl. No.	Dry weight [mg]	Growth rate of dry weight [d^-1]		Inhibition of growth rate of dry weight [%]	Yield of dry weight [mg]			Inhibition of yield of dry weight [%]
100	1	36.1		0.360	-2			33.2	-5
	2	34.6		0.354	-1			31.7	-1
	3	40.0		0.375	-7			37.1	-18
	4	35.0		0.356	-1			32.1	-2
	5	31.4		0.340	3			28.5	10
	6	37.1		0.364	-3			34.2	-9
	Mean	35.7	(-)	0.358	-2	(-)		32.8	-4
Control	1	28.1		0.324			25.2
	2	29.4		0.331			26.5
	3	35.9		0.359			33.0
	4	33.1		0.348			30.2
	5	39.8		0.374			36.9
	6	40.4		0.376			37.5
	Mean	34.4		0.352			31.5

Initial biomass dry weight was 2.9 mg per replicate

Repl. No. = Replicate number

 

 Colony Number (Plants) on Days 0 and 7

Nominal loading of thetest item [mg/L]		Replicate No.		Colony number
				0 days		7 days
100		1		3		17
		2		3		27
		3		3		21
		4		3		21
		5		3		15
		6		3		18
		Mean		3		20
Control		1		3		18
		2		3		14
		3		3		20
		4		3		14
		5		3		18
		6		3		17
		Mean		3		17

 

 Further Observations on Days 2, 5 and 7

Nominal loading of thetest item [mg/L]	Observations on day
	2	5	7
100	1	1	1
Control	1	1	1

Observations were made compared to the appearance of control colonies (plants) and vessels

1     = No observed effects

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

At the saturated solution of the test item with a nominal concentration of 100 mg/L no effects were observed on the growth rate and yield (frond and dry weight) of Lemna gibba.
All effect values are given based on the nominal loading levels of the test item.

Executive summary:

The effects of test item ) on the growth of the monocotyledon species Lemna gibba was determined according to the principles of OECD 221.

A semi-static exposure over 7 days was carried out. Based on a preliminary test a saturated solution with a nominal concentration of 100 mg/L was tested. Six replicates were investigated for the limit concentration and for the control. Frond numbers were assessed on days 0, 2, 5 and 7. Environmental parameters light, temperature and pH-value were within the acceptable limits.The validity criteria of the test guideline were fulfilled.

 

No analytical monitoring of the test item concentration was carried out because a measured concentration below the system quantification limit of the analytical method (0.4 mg/L of the test item) was determined during the non GLP preliminary range finding test in the saturated solution . This was also expected for the definitive study with Lemna gibba.

All effect values are given based on the nominal loading levels of the test item.

 

 

NOEC-, LOEC-, EC-Values and 95 % Confidence Intervals of the test item after 7 Days based on nominal loading of the test item [mg/L]

Frond number		Dry weight
Growth Rate Inhibition [mg/L]
NOEC	100	NOEC	100
LOEC	> 100	LOEC	>100
ErC10	>100	ErdwC10	>100
ErC20	>100	ErdwC20	>100
ErC50	>100	ErdwC50	>100
Yield Inhibition [mg/L]
NOEC	100	NOEC	100
LOEC	>100	LOEC	>100
EyC10	>100	EydwC10	>100
EyC20	>100	EydwC20	>100
EyC50	>100	EydwC50	>100