Trisodium bis[4-[4,5-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-3-methyl-5-oxo-1H-pyrazol-1-yl]benzene-1-sulphonato(3-)]cobaltate(3-)

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 29th to April 24th, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For determination of the test item concentrations, samples were taken from each concentration level and control 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 without use of any solubilizing agent. A stock solution was first prepared by dissolving an amount of 0.2254 g test item in 1500 mL dilution water (20X AAP medium, see 5.4), resulting a nominal concentration of 150 mg/L. This stock solution was handled by ultrasonic bath for 8 x 10 minutes at 30 °C thereafter to achieve the test item stock solution. The test solutions of the subsequent lower test concentrations were prepared by the appropriate dilution of this stock solution.
The test solutions were prepared in the testing laboratory just before introduction of Lemna colonies (start of the experiment).

Preparation of test solutions
Nominal concentration (mg/l) Test Item Stock Solution (TISS) (ml) 20X AAP medium (ml)
150 600 -
75 300 q.s. ad 600
37.5 150 q.s. ad 600
18.75 75 q.s. ad 600
9.375 37.5 q.s. ad 600
q.s. ad = quantum sufficiat ad (a sufficient quantity to make)

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: Duckweed (Lemna gibba)
- Source (laboratory, culture collection): Friedrich Schiller Universität, Institut für Allgemeine Botanik und Pflanzenphysiologie, Jena, Germany. Organisms were in-house cultureed and kept under axenic conditions; preculture was prepared 7 days before the tests.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

24.3 - 25.2°C in the test vessels

pH:

7.83 - 8.88

Nominal and measured concentrations:

Nominal concentrations: 9.375, 18.75, 37.5, 75 and 150 mg/L.

Details on test conditions:

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

GROWTH MEDIUM
- Standard medium used: 20X AAP Medium.

OTHER TEST CONDITIONS
- Photoperiod: the test vessels were placed randomly on a tray, illuminated continuously.
- Light intensity and quality: between 6500-10000 lux (8293 lux (SD: 400)) using fluorescent light tubes (with a spectral range of 400-700 nm).

EFFECT PARAMETERS MEASURED
- Determination of frond number: at the end of the test fronds were counted in each test vessel.
- Dry weight determination: every colonies, fronds and root fragments of each test vessel were collected and the dry weight 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.

RANGE-FINDING STUDY - I
- Test concentrations: 0.1, 1, 10, 100 mg/l
- Test conditions: 3 replicates per test item treated group (containing 11 fronds in total per test vessel) for a period of 7 days. A concurrent control was run (with 3 replicates).
- Results used in the evaluation:
Nominal conc. (mg(l) Untreated control 0.1 1 10 100
Average number of fronds (day 7) 138.67 132.67 110.33 106.00 35.67
Growth rates (µ) (0-7d) 0.362 0.355 0.328 0.323 0.167
% Inhibition of µ (0-7d) - 2.01 9.36 10.68 53.89

RANGE-FINDING STUDY - II
- Test concentrations: 1, 10, 50, 100, 150 mg/l
- Test conditions: 3 replicates per test item treated group (containing 11 fronds in total per test vessel) for a period of 7 days. A concurrent control was run (with 3 replicates).
- Results used in the evaluation:
Nominal conc. (mg(l) Untreated control 1 10 50 100 150
Average number of fronds (day 7) 168.67 168.67 152.33 91.33 62.33 59.33
Growth rates (µ) (0-7d) 0.389 0.390 0.375 0.302 0.248 0.240
% Inhibition of µ (0-7d) - -0.19 3.67 22.43 36.36 38.26

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-1.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (tested at least twice a year)

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

119.829 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

34.018 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

9.375 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

18.75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

GROWTH RATE
Growth rate based on frond number
NOErC (7d): 9.375mg/l, nominal; LOErC (7d): 18.75 mg/l, nominal
ErC50 (7d):119.829 mg/l, nominal

Growth rate based on dry weight
NOErC (7d):18.75 mg/l, nominal; LOErC (7d): 37.5 mg/l, nominal
ErC50 (7d): 103.693 mg/l, nominal


YIELD
Yeld based on frond number
NOEyC (7d): 9.375 mg/l, nominal; LOEyC (7d): 18.75 mg/l, nominal
EyC50 (7d): 34.018 mg/l, nominal

Yeld based on dry weight
NOEyC (7d): 18.75 mg/l, nominal; LOEyC (7d): 37.5 mg/l, nominal
EyC50 (7d): 37.5 mg/l, nominal

MEASURED CONCENTRATIONS
The measured concentrations remained within ± 20 % of the nominal at the start and at the end of the test. Therefore, the biological results and endpoints are based on the nominal concentrations..

Results with reference substance (positive control):

ErC50 (7d):7.624 mg/l, based on frond numbers
ErC50 (7d): 6.497 mg/l, based on dry weight
EyC50 (7d): 5.187 mg/l, based on frond numbers
EyC50 (7d): 5.136 mg/l, based on dry weight

Growth rates (µ) and percentage inhibition of µ

Concentration[mg/l]	Growth rate (r) and % inhibition of r, based on frond number		Growth rate (r) and % inhibition of r, based on dry weight
	r	% Ir	r	% Ir
Control	0.3867	-	0.3448	-
9.375	0.3685	4.69	0.3308	4.09
18.75	0.3127*	19.12	0.2700	21.70
37.5	0.2887*	25.34	0.2446*	29.06
75	0.2350*	39-23	0.1912*	44.55
150	0.1772*	54.16	0.1554*	54.93

 Growth rate (r) and % inhibition of r, based on frond number

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

 Growth rate (r) and % inhibition of r, based on dry weight

*: statistically significantly different from the control (Kruskal-Wallis Test; α = 0.05)

Yield rates (y) and percentage inhibition of y

Concentration[mg/l]	Yield and % inhibition of yield, based on frond number		Yield and % inhibition of yield, based on dry weight
	y	% Iy	y	% Iy
Control	154.17	-	0.00698	-
9.375	134.67	12.65	0.00624	10.58
18.75	87.33*	43.35	0.00385	44.88
37.5	72.33*	53.08	0.00312*	55.29
75	46.00*	70.16	0.00192*	72.44
150	27.33*	82.27	0.00135*	80.61

 Yield and % inhibition of yield, based on frond number

*: statistically significantly different from the control (Dunnett’s Test, α = 0.05)

Yield and % inhibition of yield, based on dry weight

*: statistically significantly different from the control (Kruskal-Wallis Test; α = 0.05)

Observations 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
	Symptoms	Degree of change	Symptoms	Degree of change	Symptoms	Degree of change
Control	–	–	–	–	–	–
9.375	–	–	–	–	–	–
18.75	–	–	Chlorosis	*	Chlorosis	*
37.5	Chlorosis	*	Chlorosis	*	Chlorosis	*
75	Chlorosis Frond size Root length	* * *	Chlorosis Frond size Root length	* * *	Chlorosis Frond size Root length	** ** *
150	Chlorosis Frond size Root length	* * *	Chlorosis Frond size Root length	* * *	Chlorosis Frond size Root length Necrosis	** ** ** *

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

*: slight, **: medium, ***: strong change

Abbreviations

Root length: decrease of the root length

Chlorosis: Colour loss of the plants

Frond size: decrease of frond size

Necrosis: Dead (i.e. white or water-soaked) frond tissue

Validity criteria fulfilled:

yes

Remarks:

doubling time of frond number in the control was 1.79 days

Conclusions:

ErC50 (7d) growth rate: 119.829 mg/l (nominal), based on frond number
EyC50 (7d) yield: 34.018 mg/l (nominal), based on frond number

Executive summary:

The test item was assessed for Lemna sp. as per OECD guideline 221.

Lemna gibba was exposed to the nominal concentration of 9.375, 18.75, 37.5, 75 and 150 mg/L of the test item over a period of 7 days (168 hours) under static conditions.

For determination of the test item concentrations, samples were taken from each concentration level and control at the start and at the end of the test and were determined using HPLC method with VIS detection.

The measured concentrations remained within ± 20 % of the nominal at the start and at the end of the test. Growth and inhibition of growth in the test solutions were compared with that in the untreated control.

The ErC50 and EyC50 values for growth rate and yield for frond number were found to be 119.829 and 34.018 mg/l, respectively, with 95 % confidence limits for growth rate of 101.759 and 146.225 mg/l and 27.774 and 41.352 mg/l 95 % confidence limits for yield.

The ErC50 and EyC50 values for growth rate and yield for dry weight was found to be 103.693 and 33.484 mg/l, respectively; with 95 % confidence limits for growth rate of 89.220 and 124.090 mg/l and 95 % confidence limits for yield of 27.240 and 40.772 mg/l.

The 7-days NOEC related to both growth rate and yield (based on frond number and dry weight) was determined to be 9.375 mg/l and the LOEC was determined to be 18.75 mg/l.

Conclusion

ErC50 (7d) = 119.829 mg/l (nominal), based on frond number

EyC50 (7d) = 34.018 mg/l (nominal), based on frond number

Description of key information

ErC50 (7d): 119.829 mg/l (nominal), based on frond number

EyC50 (7d): 34.018 mg/l (nominal), based on frond number

Key value for chemical safety assessment

EC50 for freshwater plants:

119.829 mg/L

Additional information

The test item was assessed for Lemna sp. as per OECD guideline 221.

Lemna gibba was exposed to the nominal concentration of 9.375, 18.75, 37.5, 75 and 150 mg/L of the test item over a period of 7 days (168 hours) under static conditions.

For determination of the test item concentrations, samples were taken from each concentration level and control at the start and at the end of the test and were determined using HPLC method with VIS detection.

The measured concentrations remained within ± 20 % of the nominal at the start and at the end of the test. Growth and inhibition of growth in the test solutions were compared with that in the untreated control.

The ErC50 and EyC50 values for growth rate and yield for frond number were found to be 119.829 and 34.018 mg/l, respectively, with 95 % confidence limits for growth rate of 101.759 and 146.225 mg/l and 27.774 and 41.352 mg/l 95 % confidence limits for yield.

The ErC50 and EyC50 values for growth rate and yield for dry weight was found to be 103.693 and 33.484 mg/l, respectively; with 95 % confidence limits for growth rate of 89.220 and 124.090 mg/l and 95 % confidence limits for yield of 27.240 and 40.772 mg/l.

The 7-days NOEC related to both growth rate and yield (based on frond number and dry weight) was determined to be 9.375 mg/l and the LOEC was determined to be 18.75 mg/l.