Sodium 4-[[4-[(4-hydroxy-2-methylphenyl)azo]phenyl]amino]-3-nitrobenzenesulphonate

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:

From June 30th to July 07th, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

adopted 23rd March, 2006

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Acid Orange 067

IUPAC Name:

Acid Orange 067

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sample collection: samples were taken from each concentration level and control at the start and at the end of the test.
- Replicates: 3 replicate (5 ml per replicate) taken from the test concentrations and 6 replicates (5 ml per replicate) from the control.

Test solutions

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 an amount of 1.20 g test item in 1200 ml dilution water (20X AAP medium), resulting a nominal concentration of 1000 mg/l. This solution was handled by ultrasonic bath for 20 minutes thereafter stirred rigorously for a period of 24 hours to achieve an equilibrated concentration. The dense, dark orange resulting suspension was then filtrated through a membrane filter (0.45 µm; Millipore Express® PLUS membrane*) to separate the possible non-dissolved test material. During filtration strong coloration of the membrane was observed due to undissolved test item residue, resulting in a light orange test item stock solution. The test solutions of the subsequent lower test concentrations were prepared by appropriate dilution of this stock solution.

Test organisms

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; Date of arrival totesting laboratory was 05 October 2015.
- Preculture: in-house culture; 7 days before testing, sufficient colonies are transferred from the stock culture aseptically into fresh sterile medium and cultured under the conditions of the test prior to beginning the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Test temperature:

24.0 – 25.9 °C in the climate chamber
24.3 – 24.5 °C in the test vessels

pH:

7.85 – 9.26

Nominal and measured concentrations:

12.8, 32, 80, 200, 500 and 1000.0 mg/l, nominal.
0.01, 0.02, 0.06, 0.14, 0.30 and 0.69 mg/l, calculated by extrapolation from the geometric mean.

Details on test conditions:

TEST SYSTEM
- Test vessel: 400 ml Petri dishes-covered glass beakers.
- Fill volume: beakers were filled with 160 ml testing solutions.
- No. of colonies per vessel: two colonies.
- No. of fronds per colony: two colonies with four and one colony with three fronds per vessel.
- 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.
- No. of vessels per concentration: 3 replicates.
- No. of vessels per control: 6 replicates.

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: 20X AAP medium, prepared in the testing laboratory, as described into the OECD guideline 221.
- Filtration: the medium was filtered through a 0.22 µm membrane filter into sterile container.

OTHER TEST CONDITIONS
- Adjustment of pH: pH was adjusted to 7.5 ± 0.1 with 1 N HCl.
- Photoperiod: test vessels were illuminated continuously.
- Light intensity and quality: light intensity between 6500-10000 lux using fluorescent light tubes (with a spectral range of 400-700 nm). 7952 lux was the mean value during the experiment.

EFFECT PARAMETERS MEASURED
- Frequency of observation: number and appearance of fronds were determined during the 168-hour test on the 3rd, 5th and 7th days.
- Determination of biomass: effects of the test item on final biomass were also assessed based on determination of dry weight at the beginning and at the end of the study.

MEASUREMENTS
- Temperature: checked at the beginning of the study and every 24 hours (in a surrogate flask filled with water in the climate chamber). In addition, the temperature was continuously measured (with a min/max thermometer) within the climate chamber during the experimental period.
- pH: pH was checked at the start and at the end of the test in the test concentrations and the control. The pH of the control medium did not increase by more than 1.5 units during the test.
- Light intensity: the light intensity was checked and recorded at the start of the test at the position occupied by test containers. 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.

PRELIMINARY TEST
- No. of vessels per concentration: 2 replicates per test item treated group (containing 11 fronds in total per test vessel).
- No. of vessels per control: 3 replicates.
- Duration: 7 days.
- Concentrations: 0.1, 1, 10, 100, 500, 1000 mg/l, nominal; 100, 500 and 1000 mg/l were measured to be 0.2, 1.8 and 3.8 mg/l, respectively.
- Choice for statit test: during analytical method validation the test item in the clear solvent phase of the saturated stock solution resulted to be stable for the duration of the experiment in 20X AAP medium; therefore the substance is not expected to undergo to transformation/degradation phenomena. However, during the preliminary test, a decrease of test item concentrations was recorded, due to adsorption on test organism. Because the renewal of the solution could determine higher amounts of test item adsorbed to organisms (with a possible consequently negative impact due to “physical” interference and not due to a real toxicity), a static system was preferred.

VALIDITY
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 an 7-fold increase in 7 days and an average specific growth rate of 0.275 d-1.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

7 d

Dose descriptor:

EC50

Remarks:

graphically estimated

Effect conc.:

> 0.69 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

7 d

Dose descriptor:

EC50

Remarks:

graphically estimated

Effect conc.:

0.3 - 0.69 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

No acute toxicity (i.e. ErC50) was recorded up to the higher concentration reached in the test. The decrease of the measured concentrations during the experiment was due to the test substance adsorption to the lemna roots.

The average specific growth rate was statistically significantly different from the control group in the concentrations of 0.14, 0.30 and 0.69 mg/l (calculated) based on frond number and dry weight (Dunnett’s test (2-sided, α = 0.05)).
The 7-d ErfnC50 was determined to be higher than 0.69 mg/l.
The 7-d ErdwC50 was determined to be higher than 0.69 mg/l.
The effects observed at 500 and 1000 mg/l regard the 27 and 35 % of population, respectively. Because of the adsorption occurrance, effects may be due to a physical impact. Same effects were observed in the prelimnary test, in which flattening of the effects curve shape was evident.

Yield (0-7d) was statistically significantly different from the control group in the four highest concentrations of 0.06, 0.14, 0.30 and 0.69 mg/l (calculated) based on frond number and in the three highest test concentrations of 0.14, 0.30 and 0.69 mg/l (calculated) based on dry weight (Dunnett’s test (2-sided, α = 0.05)).
The 7-d EyfnC50 was determined to be between 0.30 and 0.69 mg/l The 7-d EydwC50 was determined to be between 0.30 and 0.69 mg/l.

MEASURED CONCENTRATIONS
Nominal concentrations of 12.8, 32, 80, 200, 500 and 1000 mg/l were investigated in the main study. Tested solutions appeared clear, without any suspended matter or precipitation.
As previously mentioned, after stirring, the suspension that was filtrated, thus most of the test item was lost already at the start of the experiment, the further decrease of test item concentration measured was due to adsorption onto the roots of the plants.
The measured concentrations deviated more than 20 % from the nominal at the start and at the end of the test. Only the two highest test concentrations were over the quantitation limit therefore the geometric mean of this measured concentrations were calculated to determine exposure concentrations. The further test item concentrations were calculated by extrapolation from the geometric mean of the highest measured test item concentration. The corresponding calculated concentrations were the followings: 0.01, 0.02, 0.06, 0.14, 0.30 and 0.69 mg/l

PRELIMINARY RANGE-FINDING TEST
Effects with same magnitude were observed at 500 and 1000 mg/l and this let to suspect of a physical impact due to substance absorption (because was not a dose-related) and the possible achievement of a “satured” level (with the consequent flattening of the effects curve shape).

Results with reference substance (positive control):

3,5-dichlorophenol is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study with reference item 3,5-dichlorophenol was: 03 – 10 February 2017.
Endpoints of this study were:
EyC50 (7 day, based on frond numbers): 6.42 mg/l;
ErC50 (7 day, based on frond numbers): 7.76 mg/l;
EyC50 (7 day, based on dry weight): 6.08 mg/l;
ErC50 (7 day, based on dry weight): 7.71 mg/l.

Any other information on results incl. tables

Growth rates (r) and percentage inhibition of r based on frond number

Concentration[mg/l]		Growth rate (r) and % inhibition of r
Nominal	Calculated	0–7 d (based on frond number)
		r	% Ir
Control	Not detected	0.334	-
12.8 (TISS/78.125)	0.01	0.334	-0.09**
32 (TISS/31.25)	0.02	0.330	1.06
80 (TISS/12.5)	0.06	0.323	3.34
200 (TISS/5)	0.14	0.304*	8.95
500 (TISS/2)	0.30	0.242*	27.42
1000 (TISS)	0.69	0.218*	34.75

TISS: Test Item Stock Solution

*: statistically significantly different from the control (Dunnett’s Test; 2-sided; α = 0.05)

**: negative value indicates increase in comparison to the control. It is calculated as 0.00 in the statistics.

Growth rates (r) and percentage inhibition of r based on dry weight

Concentration[mg/l]		Growth rate (r) and % inhibition of r
Nominal	Calculated	0–7 d (based on dry weight)
		r	% Ir
Control	Not detected	0.36467	-
12.8 (TISS/78.125)	0.01	0.35962	1.38
32 (TISS/31.25)	0.02	0.35450	2.79
80 (TISS/12.5)	0.06	0.34334	5.85
200 (TISS/5)	0.14	0.32118*	11.93
500 (TISS/2)	0.30	0.28312*	22.36
1000 (TISS)	0.69	0.25317*	29.60

TISS: Test Item Stock Solution

*: statistically significantly different from the control (Dunnett’s Test; 2-sided; α = 0.05)

Yield (y) and percentage inhibition of yield based on frond number

Concentration[mg/l]		Growth rate (r) and % inhibition of r
Nominal	Calculated	0–7 d (based on dry weight)
		r	% Ir
Control	Not detected	102.83	-
12.8 (TISS/78.125)	0.01	103.00	-0.16**
32 (TISS/31.25)	0.02	100.00	2.76
80 (TISS/12.5)	0.06	94.33*	8.27
200 (TISS/5)	0.14	81.33*	20.91
500 (TISS/2)	0.30	49.00*	52.35
1000 (TISS)	0.69	39.67*	61.43

TISS: Test Item Stock Solution

*: statistically significantly different from the control (Dunnett’s Test; 2-sided; α = 0.05)

**: negative value indicates increase in comparison to the control. It is calculated as 0.00 in the statistics.

Yield (y) and percentage inhibition of yield based on dry weight

Concentration[mg/l]		Growth rate (r) and % inhibition of r
Nominal	Calculated	0–7 d (based on dry weight)
		r	% Ir
Control	Not detected	0.00755	-
12.8 (TISS/78.125)	0.01	0.00723	4.19
32 (TISS/31.25)	0.02	0.00699	7.46
80 (TISS/12.5)	0.06	0.00641	15.14
200 (TISS/5)	0.14	0.00537*	28.87
500 (TISS/2)	0.30	0.00397*	47.46
1000 (TISS)	0.69	0.00309*	59.03

TISS: Test Item Stock Solution

*: statistically significantly different from the control (Dunnett’s Test; 2-sided; α = 0.05)

Observation during the test

Symptoms, changes of Lemna gibba plants observed during the test

Concentration [mg/l]		3^rdday of the experiment		5^thday of the experiment		7^thday of the experiment
Nominal	Measured	Symptoms	Degree of change	Symptoms	Degree of change	Symptoms	Degree of change
Control	Not detected	–	–	–	–	–	–
12.8 (TISS/78.125)	0.01	–	–	–	–	–	–
32 (TISS/31.25)	0.02	–	–	–	–	–	–
80 (TISS/12.5)	0.06	–	–	–	–	–	–
200 (TISS/5)	0.14	–	–	–	–	Root length Chlorosis	* *
500 (tISS/2)	0.3 (mean measured)	Root length Chlorosis	* *	Root length Root morphology Chlorosis Frond gibbosity	* * * *	Root length Root morphology Chlorosis Frond gibbosity	* * ** *
1000 (TISS)	0.69 (mean measured)	Root length Chlorosis Frond gibbosity	** ** *	Root length Root morphology Chlorosis Frond gibbosity	** * ** **	Root length Root morphology Chlorosis Frond gibbosity Frond size	** * ** ** *

Legend:

TISS: Test Item Stock Solution

"–": the plants were healthy, there was not any symptom observed

*: weak, **: middle, ***: strong change

Abbreviations:

- Root length: Decrease of the root length

- Root morphology: Morphological change of roots

- Chlorosis: Colour loss of the plants

- Frond gibbosity: Gibbosity of fronds

- Frond size: Decrease of frond size

MEASURED CONCENTRATIONS

Nominal concentration [mg/l]	Measured concentration [mg/l]			Calculated concentration [mg/l]
	Start	End	Geometric mean
Control	Not detected	Not detected	-	-
12.8 (TISS/78.125)	<LOQ*	<LOQ*	-	0.01
32 (TISS/31.25)	<LOQ*	<LOQ*	-	0.02
80 (TISS/12.5)	<LOQ*	<LOQ*	-	0.06
200 (TISS/5)	<LOQ*	<LOQ*	-	0.14
500 (TISS/2)	0.35	0.24	0.30	0.30
1000 (TISS)	0.72	0.65	0.69	0.69

TISS: Test Item Stock Solution

*below of quantification limit

PRELIMINARY RANGE-FINDING TEST

Nominal concentrations [mg/l]	Untreated control	0.1	1	10	100 (measured: 0.2)	500 (measured: 1.8)	1000 (measured: 3.8)
Average number of fronds (day 7)	57	55.5	55.5	55.5	45.5	35.5	35
Growth Rates (µ)[0-7d]*	0.235	0.231	0.231	0.231	0.202	0.167	0.165
% Inhibition of µ[0-7d]	–	1.62	1.72	1.62	13.86	28.82	28.49

* Average daily growth rate

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The doubling time of frond number in the control was 2.08 day. The validity criterion was within acceptable limit and therefore the study can be considered as valid.

Conclusions:

No acute toxicity (i.e. ErC50) was recorded up to the higher concentration reached in the test. No toxic effect was observed up to the limit of water solubility in the Lemna test medium.

Executive summary:

The effect of test item was assessed in duckweed, Lemna gibba, over an exposure period of 7 days, following the testing procedures outlined into the OECD guideline 221. Based on the results of two non-GLP preliminary tests, the nominal test item concentrations included in the study were 12.8, 32, 80, 200, 500 and 1000 mg/l. The measured concentrations deviated more than 20 % from the nominal at the start and at the end of the test. Only the two highest test concentrations were over the quantitation limit therefore the geometric mean of this measured concentrations were calculated to determine exposure concentrations. The further test item concentrations were calculated by extrapolation from the geometric mean of the highest measured test item concentration. The corresponding calculated concentrations were the followings: 0.01, 0.02, 0.06, 0.14, 0.30 and 0.69 mg/l; biological results and endpoints are based on the measured and calculated concentrations.

All validity criteria were met and therefore the study can be considered as valid.

The 7-d ErC50, based on frond number, was determined to be higher than 0.69 mg/l; the 7-d ErC50, based on dry weight, was determined to be higher than 0.69 mg/l.

The effects observed at 500 and 1000 mg/l regard the 27 and 35 % of population, respectively. Because of the adsorption occurrance, effects may be due to a physical impact. Same effects were observed in the prelimnary test, in which flattening of the effects curve shape was evident.

Yield (0-7d) was statistically significantly different from the control group in the four highest concentrations of 0.06, 0.14, 0.30 and 0.69 mg/l (calculated) based on frond number and in the three highest test concentrations of 0.14, 0.30 and 0.69 mg/l (calculated) based on dry weight (Dunnett’s test (2-sided, α = 0.05)). The 7-d EyC50, based on frond number, was determined to be between 0.30 and 0.69 mg/l The 7-d EyC50, based on dry weight, was determined to be between 0.30 and 0.69 mg/l.

No acute toxicity (i.e. ErC50) was recorded up to the higher concentration reached in the test. The decrease of the measured concentrations during the experiment was due to the test substance adsorption to the lemna roots.

Conclusion

No acute toxicity (i.e. ErC50) was recorded up to the higher concentration reached in the test.