Sodium 2-[[4-(cyclohexylamino)-9,10-dihydro-9,10-dioxo-1-anthryl]amino]-5-ethoxybenzenesulphonate

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

February from 10th to 17th, 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The read across approach is detailed into the document attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

adopted 23th March 2006

Deviations:

yes

Remarks:

temperature ranged between 18.4 and 26.4°C, which is out of the required range of 24 ± 2 °C; the deviation did not impact the test results

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analyses of the test concentrations were conducted at the beginning of the test, as well as after 3, 5 and 7 days of exposure.

Details on test solutions:

Since the test item resulted to be soluble, the test solutions were prepared by respective dilutions of a stock solution (test item dissolved in SIS Medium) with SIS Medium.

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: duckweed.
- Source: exponential growing plant monoculture of Lemna minor, from Umweltbundesamt, FGIII 2.5, Überwachungsverfahren Abwasserentsorgung, Schichauweg 58, D-12307 Berlin.

CULTIVATION
- Cultivation: all-glass vessel containing sterile Swedish standard-Medium
- Illumination: continuous (6500–10000 lux) from Osram Fluora L18W77 (Osram AG, Winterthur, Switzerland) and CH Lighting F18T8/6500K EUP (CH Lighting CO., Ltd., China)
- Temperature: 24 ± 2 °C
- Control of sensitivity: yearly, with 3,5-dichlorophenol.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

The temperature ranged between 18.4 and 26.4 °C (i.e. 6.0 °C variation), which is out of the required range of 24 ± 2 °C. Since the test vessels were randomly replaced, since all test vessels were exposed to the same temperature, since the toxicity is calculated based on the blank control, and since the growth criterion in the control was fulfilled without a too large variation, this deviation is not expected to have a significant impact on the outcome of the study.
The test item did not show any signs of precipitation, at any of the test concentrations.

pH:

The pH value in the control drifted by 0.6 units during the whole test period, which is thus in the range allowed by the guideline (required: not more than 1.5).

Nominal and measured concentrations:

611, 193, 61.1, 19.3 and 6.11 mg/l nominal concentration of the test item, corresponding to 500, 158.1, 50.0, 15.8 and 5.00 mg/l of the active ingredient

Details on test conditions:

TEST SYSTEM
- Test vessel: 400 ml beakers, all-glass, with 200 ml of test medium.
- Type of cover: the beakers were covered with black paper up until the 200 ml mark to ensure that illumination comes only from above and not from the sides.
- Incubation: beakers were incubated on a black non-reflecting surface.
- No. of frond per plant: 2-4 fronds per plant.
- No. of fronds per replicate: 9-12 fronds per replicate.
- No. of vessels per concentration: three replicates.
- No. of vessels per control : six replicates.

GROWTH MEDIUM
- Standard medium used: yes; Swedish standard-Medium.

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: Swedish standard-Medium, prepared with ultra-pure water.
- Adjustment of pH: the pH of the SIS Medium was adjusted before the test to pH 6.5 ± 0.2.

OTHER TEST CONDITIONS
- Photoperiod: continuous light.
- Light intensity and quality: the light intensity was 7500-8250 lux (mean 7939 lux; max. variation ± 6 %) at the start of the test and 6950-8340 lux (mean 7663 lux, max. variation ± 9 %) at the end of the exposure period.
- Homogeneity: the light homogeneity was in the required ±15 % range.

EFFECT PARAMETERS MEASURED
- Observations: the number of fronds was counted on day 0, 3, 5 and 7. Changes in plant development, e.g. in frond size, appearance, indication of necrosis, chlorosis or gibbosity, colony break-up, loss of buoyancy, root length and appearance were noted. Significant changes in the test medium (e.g.precipitation, growth of algae in the test vessel) were also noted.
- Dry weight: determined from duckweed at day 0 in six additional blank replicates (similar to those used in the test), and at day 7 in all blanks and all test vessels, after drying at 60 °C at least overnight.

CONDITIONS MEASUREMENT
- pH: determined in the combined replicate test solutions at the beginning and at the end of the test.
- Light intensity: measured at the beginning and at the end of the test over the whole test area at points the same distance from the light source as the Lemna fronds.
- Temperature: measured in an additional glass vessel containing test medium and Lemna, at least daily.

RANGE-FINDING STUDY
Prior to the definitive test a non-GLP range finding test was performed.
- Test concentrations: 10, 100 and 500 mg/l of test item active ingredient (nominal). The photometry determinations indicate that the test item concentrations were stable. Static conditions were therefore applied in the definitive test.
- Results used to determine the conditions for the definitive study: the percentages of inhibition, based on frond number were
at 10 mg/l: 3.4 % growth rate inhibition, 8.1 % yield inhibition
at 100 mg/l: 35 % growth rate inhibition, 61 % yield inhibition
at 10 mg/l: 59 % growth rate inhibition, 82 % yield inhibition

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

615 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

889 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

197 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

178 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

EFFECTS ON FROND NUMBER
With respect to growth rate inhibition, the following effects as compared to the untreated controls were observed: 39 % at 500 mg/l and 23 % at 158 mg/l. No significant effects were observed at the concentrations 50.0, 15.8 and 5.00 mg/l.
Based on these effects and the measured concentrations of the active ingredient, the median effect concentration with respect to the frond number’s growth rate (frond number ErC50) of test item to Lemna minor was calculated to be 615 mg/l (95 % confidence limits: 439–1115 mg/l). The ErC10 was 62.0 mg/l (29.6–92.3 mg/l); while the NOErC was determined to be 49.1 mg/l.

With respect to yield inhibition, the following effects as compared to the untreated controls were observed: 66 % at 500 mg/l and 46 % at 158 mg/l. No significant effects were observed at the concentrations 50.0, 15.8 and 5.00 mg/l.
Based on these effects and the measured concentrations of the active ingredient, the median effect concentration with respect to the frond number’s yield (frond number EyC50) of test item to Lemna minor was calculated to be 197 mg/l (95 % confidence limits: 141–300 mg/l). The EyC10 was 29.2 mg/l (9.26–50.6 mg/l); while the NOEyC was determined to be 49.1 mg/l.

EFFECTS ON DRY WEIGHT
With respect to growth rate inhibition, the following effects as compared to the untreated controls were observed: 34 % at 500 mg/l and 23 % at 158 mg/l. No significant effects were observed at the concentrations 50.0, 15.8 and 5.00 mg/l.
Based on these effects and the measured concentrations of the active ingredient, the median effect concentration with respect to the dry weight’s growth rate (dry weight ErC50) of test item to Lemna minor was calculated to be 889 mg/l (95 % confidence limits: 546–2429 mg/l). The ErC10 was 57.5 mg/l (22.5–91.2 mg/l); while the NOErC was determined to be 49.1 mg/l.

With respect to yield inhibition, the following effects as compared to the untreated controls were observed: 65 % at 500 mg/l and 52 % at 158 mg/l. No significant effects were observed at the concentrations 50.0, 15.8 and 5.00 mg/l.
Based on these effects and the measured concentrations of the active ingredient, the median effect concentration with respect to the dry weight’s yield (dry weight EyC50) of test item to Lemna minor was calculated to be 178 mg/l (95 % confidence limits: 124–280 mg/l). The EyC10 was 20.8 mg/l (6.01–38.0 mg/l); while the NOEyC was determined to be 49.1 mg/l.

APPEARANCE OF THE PLANTS AT THE END OF THE TEST
In the blank control the plants were healthy with dark green fronds and roots reaching to the bottom of the test vessel.
At 5.00 mg/l, the plants looked like the blanks, with dark green fronds and roots reaching to the bottom of the test vessel.
At 15.8 and 50.0 mg/l, the plants were healthy with dark green fronds and roots reaching to the bottom of the test vessel; the plants were slightly smaller compared to the blank control.
At 158 mg/l, the fronds were dark green and somehow misshapen; the size of the roots were only two thirds of the blank control and the plants were significantly smaller as compared to the blank controls.
At 500 mg/l, the plants werevery small, with plants and fronds partly blue discolored; the size of the roots were only two thirds of the blank control.

TEST CONCENTRATIONS
The determination of test concentrations showed that the test item decreased over the whole 7-day test period. The measured concentrations of the active ingredient at the beginning of the test were 514, 163, 49.3, 16.0 and 4.93 mg/l and 345, 113, 50.6, 14.3 and 4.24 mg/l (i.e. 67, 69, 103, 90 and 86 %, respectively, of the initial value) after 7 days.
Therefore, the effective concentrations (ErC50 and EyC50) were assessed based on the geometric mean (GM) of the measured concentrations of the active ingredient.

Validity criteria fulfilled:

yes

Remarks:

doubling time of frond number in the control was less than 2.5 days (60 h), corresponding to approximately a seven-fold increase in seven days and an average specific growth rate of 0.275 d-1

Conclusions:

ErC50 (7d): 615 mg/l (meas. geom mean), based on frond number
EyC50 (7d): 197 mg/l (meas. geom mean), based on frond number

Executive summary:

The inhibitory effects of test item to the duckweed Lemna minor were investigated over a period of 7 days, based on the frond number and biomass (dry weight), following the guideline OECD 221. The test solutions were prepared by respective dilutions of a stock solution in Swedish standard-Medium (SIS Medium).

The test was performed at 611, 193, 61.1, 19.3 and 6.11 mg/l nominal concentration of the test item, corresponding to 500, 158.1, 50.0, 15.8 and 5.00 mg/l of the active ingredient.

Three parallel test vessels were used for each test concentration of the test item and six vessels for the blank controls.

The test concentrations of test item during the 7-day test period were determined by photometry at the beginning of the test, as well as after 3, 5 and 7 days of exposure. These analyses confirmed the right dosage of the test item, and showed that the concentrations of the loadings decreased over the whole 7-day test period after 7 days and did not remain within 80-120 % range of the initial value (67, 69, 103, 90 and 86 %, respectively, of the initial value). Therefore, the effective concentrations (ErC50 and EyC50) were assessed based on the geometric mean (GM) of the measured concentrations of the active ingredient.

The two endpoints frond number and biomass (dry weight) were investigated at days 3, 5 and 7, and each of them were assessed as growth rate and yield.

The results of the toxicity assessment of to the duckweed Lemna minor are summarized below, showing ECx values based on the measured concentrations of active ingredient:

ErC50 (7d): 615 mg/l (meas. geom mean), based on frond number

ErC50 (7d): 889 mg/l (meas. geom mean), based on dry weight

EyC50 (7d): 197 mg/l (meas. geom mean), based on frond number

EyC50 (7d): 178 mg/l (meas. geom mean), based on fdry weight

The No Observed Effect Concentration was 49.1 mg/l in all the cases (determined by Dunnett's test).

Conclusion

ErC50 (7d): 615 mg/l (meas. geom mean), based on frond number

EyC50 (7d): 197 mg/l (meas. geom mean), based on frond number

Reference 2

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From November 16th to 23rd, 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The read across approach is detailed into the document attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

23 March, 2006

Qualifier:

according to guideline

Guideline:

other: EU Method C.26 (Lemna sp. Growth Inhibition Test)

Version / remarks:

24 August 2009

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 23 (Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures)

Version / remarks:

September 2000

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples were taken from each concentration level and control.
- Sampling timing: samples were taken at the start and at the end of the test.

Vehicle:

no

Details on test solutions:

The test solutions used in the test were prepared by mechanical dispersion. A stock solution was first prepared by dissolving 0.2402 g of test item in 1501.25 ml dilution water (20X AAP medium). The stock solution (160 mg/l nominal concentration) was handled in ultrasonic bath for approx. 10 minutes thereafter stirred rigorously during approx. 24 hours. The resulting saturated solution was then filtrated through a membrane filter (0.45 µm pore size). The test solutions of the subsequent lower test concentrations were prepared by appropriate diluting of this clear solvent phase of the saturated stock solution. The test solutions were freshly prepared in the testing laboratory just before introduction of the test organisms (start of the test).

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: duckweed.
- Source: Friedrich Schiller Universität, Institut für Allgemeine Botanik und Pflanzenphysiologie, Jena, Germany.
- Preculture: 7 days before testing sufficient colonies were transferred from the stock culture aseptically into fresh sterile medium and cultured under the conditions of the test prior to beginning the test.
- Initial frond number: the initial frond number in the test cultures was 11. The number of colonies and fronds was identical in each test vessel.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

22.1 - 24.8 °C in the climate chamber
23.0 - 23.6 °C in the test vessels

pH:

7.87 - 8.59

Nominal and measured concentrations:

5.0, 10.0, 20.0, 40.0, 80.0 and 160 mg/l, nominal (1.29, 2.54, 5.49, 10.97, 23.69 and 32.70 mg/l, geom. means, respectively).

Details on test conditions:

TEST SYSTEM
- Incubation environment: containers were kept during the test in a climate chamber with controlled environmental conditions.
- Test vessel: 400 ml glass beakers were filled with 160 ml testing solutions.
- Type of cover: covered by glass petri dishes.
- No. of colonies per vessel: three colonies.
- No. of fronds per colony: two Lemna gibba colonies with four and one colony with three fronds were added to each vessel.
- No. of vessels per concentration: three replicates.
- No. of vessels per control: six replicates.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 20X AAP medium.

OTHER TEST CONDITIONS
- Adjustment of pH: the pH was adjusted to 7.5 ± 0.1 with 1 N HCl. The medium was then filtered through a 0.22 µm membrane filter into sterile container. The medium was prepared one day before use to allow the pH to stabilize. The pH of the medium was checked prior to use.
- Photoperiod: continuously illuminated.
- Light intensity and quality: light intensity was 7125 lux (mean value; SD: 129 lux) using fluorescent light tubes (with a spectral range of 400-700 nm). The differences in light intensity between the measurement points (i.e. position of fronds) did not exceed ± 15 % and therefore provided equal conditions for each test culture.

EFFECT PARAMETERS MEASURED
- Fresh and dry weight determination: colonies were collected and were blotted to remove excess water and then the fresh weights were determined of the colonies. Thereafter the colonies were dried at 60 °C to a constant weight. Any root fragments were included. At the end of the test fronds were counted in each test vessel. Every colonies, fronds and root fragments of each test vessel were collected and the fresh weight and dry weights were measured in the same way as at the start of the study.
- Observations: assessment of growth and morphological changes on days 3, 5 and 7. Any change in plant development, frond size appearance, was noted as well as additional observations of root length, significant features of the test media or other abnormalities.

RANGE-FINDING STUDY
- Test system: two non-GLP preliminary range-finding tests were conducted to determine the approximate toxicity of the test item.

First range finding
- Test solution: the solution used was prepared by mechanical dispersion. 0.05 g of test item was dissolved in 500 ml dilution water (20X AAP medium) in order to obtain the concentration of 100 mg/l. This solution was placed into ultrasonic bath for 10 minutes and thereafter, was stirred for 24 hours and, thereafter, the non-dissolved test material was removed by filtration through a 0.45 µm membrane filter to obtain the saturated test solution.
- No. of vessels per concentration: three replicates (containing 11 fronds in total per test vessel).
- No. of vessels per control: three replicates.
- Test concentrations: 100 mg/l.
- Results used to determine the conditions for the definitive study: 65.23 % of inhibition, based on growth rate.

Second range finding
- Test solution: the solutions used were prepared by mechanical dispersion. 0.0601 g of test item was dissolved in 600 ml dilution water (20X AAP medium; see) in order to obtain the concentration of 100 mg/l. This stock solution was placed into ultrasonic bath for 10 minutes. The further test solutions were prepared by appropriate dilution of this stock solution.
- No. of vessels per concentration: two replicates per test item treated group (containing 11 fronds in total per test vessel).
- No. of vessels per control: three replicates.
- Test concentrations: 0.1, 1.0, 10.0 and 100.0 mg/l
- Results used to determine the conditions for the definitive study: the percentage of inhibition of growth rate were 0.95, 17.88, 56.75 and 110.32 % at the concentrations of 0.1, 1.0, 10.0 and 100.0 mg/l, respectively.

ACCEPTANCE CRITERIA
For the test to be valid, the doubling time of frond number in the control must be less than 2.5 days (60 h), corresponding to approximately a 7-fold increase in 7 days and an average specific growth rate of 0.275/d.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

11.29 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

7.47 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

4.68 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

3.06 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

GROWTH RATE
The average specific growth rate (based on frond number) was statistically significantly different from the control group at the concentrations of 1.29, 2.54, 5.49, 10.97, 23.69 and 32.70 mg/l (Dunnett t-Test (2-sided, α =0.05)).
The average specific growth rate (based on dry weight) was statistically significantly different from the control group at the concentrations of 1.29, 2.54, 5.49, 10.97, 23.69 and 32.70 mg/l (Dunnett t-Test (2-sided, α=0.05)).
The average specific growth rate (based on fresh weight) was statistically significantly different from the control group at the concentrations of 1.29, 2.54, 5.49, 10.97, 23.69 and 32.70 mg/l (Dunnett t-Test (2-sided, α=0.05)).

NOErC (7d): < 1.29 mg/l, based on frond number, dry and fresh weight
LOErC (7d): 1.29 mg/l, based on frond number, dry and fresh weight
ErC50 (7d): 11.29 mg/l (95 % confidence limits: 9.211 – 14.089 mg/l), based on frond number
ErC50 (7d): 7.47 mg/l (95 % confidence limits: 6.058 – 9.212 mg/l), based on dry weight
ErC50 (7d): 8.61 mg/l (95 % confidence limits: 7.468 – 9.956 mg/l), based on fresh weight

YIELD
Yield (based on frond number) was statistically significantly different from the control group at the concentrations of 1.29, 2.54, 5.49, 10.97, 23.69 and 32.70 mg/l (Dunnett t-Test (2-sided, α=0.05)).
Yield (based on dry weight) was statistically significantly different from the control group at the concentrations of 1.29, 2.54, 5.49, 10.97, 23.69 and 32.70 mg/l (Dunnett t-Test (2-sided, α=0.05)).
Yield (based on fresh weight) was statistically significantly different from the control group at the concentrations of 1.29, 2.54, 5.49, 10.97, 23.69 and 32.70 mg/l (Dunnett t-Test (2-sided, α=0.05)).

NOEyC (7d): < 1.29 mg/l, based on frond number, dry and fresh weight
LOEyC (7d): 1.29 mg/l, based on frond number, dry and fresh weight
EyC50 (7d): 4.68 mg/l (95 % confidence limits: 4.025 – 5.383 mg/l), based on frond number
EyC50 (7d): 3.06 mg/l (95 % confidence limits: 2.535 – 3.605 mg/l), based on dry weight
EyC50 (7d): 3.57 mg/l (95 % confidence limits: 3.228 – 3.924 mg/l), based on fresh weight

BIOLOGICAL RESULTS
At the lowest concentration tested no effects were observed for the entire study period.
At the concentrations of 2.54 mg/l (geom. mean) decrease of the root length and decrease of frond size were observed; these effects were observed also at the higher concentrations.
Starting from the concentration of 5.49 mg/l (geom. mean) and throughout all the higher concentrations, increasing amounts of the test item were adsorbed to the roots.
Other effects observed, of which the degree of change increases among the dosages, were buoyancy, chlorosis and morphological change of roots. At the highest concentration (i.e. 32.70 mg/l, geom. mean) slight and medium necrosis of fronds was also observed at the day 3rd and 5th, respectively.

MEASURED CONCENTRATIONS
The measured concentrations deviated more than 20 % from the nominal during the experiment. The most of the substance is lost by filtration.
Therefore, all biological results are based on the geometric mean of the measured concentrations.

Results with reference substance (positive control):

EyC50 (7d): 5.000 mg/l, based on frond numbers
ErC50 (7d): 7.831 mg/l, based on frond numbers
EyC50 (7d): 5.609 mg/l, based on dry weight
ErC50 (7d): 7.722 mg/l, based on dry weight

Growth rates (µ) and percentage inhibition of µ

Measured concentration mg/l	Growth rate (r) and % inhibition of r 7 days
	(based on frond number)		(based on dry weight)		(based on fresh weight)
	r	% Ir	r	% Ir	r	% Ir
Control	0.34	-	0.37011	-	0.34192	-
1.29	0.308*	9.24	0.33018*	10.79	0.30972*	9.42
2.54	0.290*	14.56	0.28593*	22.74	0.26863*	21.43
5.49	0.257*	24.38	0.24938*	32.62	0.23404*	31.55
10.97	0.188*	44.71	0.17856*	51.76	0.17478*	48.88
23.69	0.135*	60.31	0.07283*	80.32	0.07122*	79.17
32.70	0.027*	92.09	-0.00678*	101.83	0.03638*	89.36

* statistically significantly different compared to the control values (Dunnett t-Test; 2-sided, α = 0.05)

Yield (y) and percentage inhibition of yield

Measured concentration mg/l	Yield (y) and % inhibition of yield 7 days
	(based on frond number)		(based on dry weight)		(based on fresh weight)
	y	% Iy	y	% Iy	y	% Iy
Control	108.00	0.0	0.00581	0	0.06721	0
1.29	84.33*	21.91	0.00428*	26.41	0.05219*	22.34
2.54	73.00*	32.41	0.00299*	48.55	0.03733*	44.46
5.49	55.67*	48.46	0.00222*	61.86	0.02794*	58.42
10.97	30.00*	72.22	0.00116*	79.98	0.01612*	76.02
23.69	17.33*	83.95	0.00032*	94.44	0.00447*	93.35
32.70	2.33*	97.84	-0.00002*	100.34	0.00195*	97.09

* statistically significantly different compared to the control values (Dunnett t-Test; 2-sided, α = 0.05)

Measured concentrations at the start and at the end of the test

Nominal Concentration, mg/l	Start		End
	Measured conc., mg/l	% of the nominal	Measured conc., mg/l	% of the nominal
5	1.32	26	1.27	25
10	2.33	23	2.77	28
20	5.45	27	5.53	28
40	10.2	26	11.8	29
80	23.2	29	24.2	30
160*	49.5	31	21.6	13

* A large amount of the test item was precipitated on the bottom of the test vessels after 3 days.

Validity criteria fulfilled:

yes

Remarks:

the doubling time of frond number in the control was 2.04 days (less than 2.5 days)

Conclusions:

ErC50 (7d): 11.29 mg/l (geom. mean), based on frond number
EyC50 (7d): 4.68 mg/l (geom. mean), based on frond number

Executive summary:

The phytotoxicity of test item on freshwater aquatic plant Lemna gibba was assessed in accordance with method and procedures outlined into OECD guideline 221.

In the main test, exponentially growing cultures of Lemna gibba were exposed to nominal concentrations 5.0, 10.0, 20.0, 40.0, 80.0 and 160 mg/l of the test item, over a period of 7 days in a static system under defined conditions.

The measured concentrations deviated more than 20 % from the nominal during the experiment in each concentration, therefore, biological results are based on the geometric mean of the measured test item concentrations.

The average specific growth rate, as well as yield, was statistically significantly different from the control group at the concentrations of 1.29, 2.54, 5.49, 10.97, 23.69 and 32.70 mg/l, based on on frond number, dry and fresh weight.  

NOEr/yC (7d): < 1.29 mg/l, based on frond number, dry and fresh weight

LOEr/yC (7d): 1.29 mg/l, based on frond number, dry and fresh weight

ErC50 (7d): 11.29 mg/l (95 % confidence limits: 9.211 – 14.089 mg/l), based on frond number

ErC50 (7d): 7.47 mg/l (95 % confidence limits: 6.058 – 9.212 mg/l), based on dry weight

ErC50 (7d): 8.61 mg/l (95 % confidence limits: 7.468 – 9.956 mg/l), based on fresh weight

EyC50 (7d): 4.68 mg/l (95 % confidence limits: 4.025 – 5.383 mg/l), based on frond number

EyC50 (7d): 3.06 mg/l (95 % confidence limits: 2.535 – 3.605 mg/l), based on dry weight

EyC50 (7d): 3.57 mg/l (95 % confidence limits: 3.228 – 3.924 mg/l), based on fresh weight

Conclusion

ErC50 (7d): 11.29 mg/l (geom. mean), based on frond number

EyC50 (7d): 4.68 mg/l (geom. mean), based on frond number

Description of key information

ErC50 (7d) = 10 - 100 mg/l for frond number

Key value for chemical safety assessment

EC50 for freshwater plants:

11.29 mg/L

Additional information

There are no information about the potential toxicity of Acid Blue 280 to aquatic plants, thus the available information on the structural analogues Similar Substance 01 and Similar Substance 02 were used.

The read across approach can be considered as appropriate and suitable; details about the approach are reported into the IUCLID section 13.

Data on the 2 structural analogues were evaluated in a weight of evidence approach, as both studies were reliable. A precautionary approach was followed to the purpose of classification.

Toxicity of Similar Substance 01 on Lemna gibba is tested in a 7 -day static test in 20X AAP medium. Based on the results of the preliminary experiments, nominal concentrations of 5.0, 10.0, 20.0, 40.0, 80.0 and 160 mg/L were used in the main study. As measured concentrations deviated more than 20 % from the nominal during the experiment in each concentration, biological results were based on the geometric mean of measured values.

Test concentrations were tested with 3 replicates; control group with 6 replicates. Test concentrations were prepared upon dilution of a stock solution with test medium. Assessment of growth and morphological changes were done on days 3, 5 and 7.

Test item concentrations were monitored at the start and at the end of the test, by HPLC-UV.

Effects of Similar Substance 01 were assessed as growth rate and yield based on frond number, dry weight and fresh weight.

ErC50 (7d): 11.29 mg/l (meas. geom mean), based on frond number

ErC50 (7d): 7.47 mg/l (meas. geom mean), based on dry weight

ErC50 (7d): 8.61 mg/l (meas. geom. man), based on fresh weight

EyC50 (7d): 4.68 mg/l (meas. geom mean), based on frond number

EyC50 (7d): 3.06 mg/l (meas. geom mean), based on dry weight

EyC50 (7d): 3.57 mg/l (meas. geom. man), based on fresh weight

The NOEC was < 1.29 mg/l and LOEC was 1.29 mg/l in all the cases (determined by Dunnett's test).

The inhibitory effects of Similar Substance 02 to the duckweed Lemna minor were investigated over a period of 7 days, based on the frond number and biomass (dry weight), following OECD guideline 221. Concentrations of test item during the 7 -day test period were determined by photometry; the analyses showed that the concentrations of the loadings decreased over the whole 7 -day test period and did not remain within 80-120 % range of the initial value (67, 69, 103, 90 and 86 %, respectively, of the initial value). Therefore, effective concentrations (ErC50 and EyC50) were assessed based on the geometric mean (GM) of the measured concentrations of the active ingredient.

The results of the toxicity assessment to the duckweed Lemna minor are summarized below, showing ECx values based on the measured concentrations of active ingredient:

ErC50 (7d): 615 mg/l (meas. geom mean), based on frond number

ErC50 (7d): 889 mg/l (meas. geom mean), based on dry weight

EyC50 (7d): 197 mg/l (meas. geom mean), based on frond number

EyC50 (7d): 178 mg/l (meas. geom mean), based on dry weight

The NOEC was 49.1 mg/l in all the cases (determined by Dunnett's test).