Trisodium 3-[[4-[[6-(anilino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]-5-methoxy-o-tolyl]azo]naphthalene-1,5-disulphonate

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Remarks:

lemna species

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 November 2016 to 14 February 2017

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)

Principles of method if other than guideline:

pH in controls increased from 6.7 to 9.9-10.1 (pH increase in the lower test concentrations also > 1.5)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: at 0, 1.0, 3.2, 10, 32 and 100 mg/L on day 0 and 7 (range finding at 1, 10 and 100 mg/L on day 0, 3 and 7)
- Sampling method: freshly prepared bulk test preparations on day 0 and old pooled samples on day 7
- Sample storage conditions before analysis: frozen

Vehicle:

no

Details on test solutions:

directly dissolved in culture medium

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: duckweed
- Strain: Lemna minor
- Source (laboratory, culture collection): Canadian Phycological Culture Centre, University of Waterloo, Ontario, Canada.

ACCLIMATION
- Acclimation period: at a temperature of 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) during at least 7 days
- Culturing media and conditions (same as test or not): yes (Swedish Standard (SIS) Lemna growth medium)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

24 ± 1 ºC

pH:

on day 0: 6.7
on day 7: >= 8.7 in controls and at 1 and 3.2 mg/L; 7.3-8.1 at 10, 32 and 100 mg/L

This increase was considered to be due to the amount of carbon dioxide required by the large number of colonies present exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 7 Days was in excess of that recommended in the Test Guidelines (1.5 pH units after 7 Days). This was considered to have had no adverse effect on the results of the study given that the control validity criterion was met.

Nominal and measured concentrations:

nominal:
pre-test: 0, 1, 10 and 100 mg/L
main test: 0, 1.0, 3.2, 10, 32 and 100 mg/L
measured:
pre-test: on day 0, 3 (old) and 7 (old): 1 mg/L 25-35%, 10 mg/L 30-32%, 100 mg/L 81-95% of nominal
main tests at 0, 1.0, 3.2, 10, 32 and 100 mg/L on day 0: on day 7 (old):

Details on test conditions:

TEST SYSTEM
- Test vessel: glass conical flasks (500 mL) each containing 250 mL of test preparation
- Aeration: no
- Agitation: no
- Renewal rate of test solution: main study static test (pre-test semistatic with renewal on day 3)
- No. of fronds per colony: 3 colonies (10 fronds)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, Swedish Standard (SIS) Lemna growth medium

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continously
- Light intensity and quality:ca 7000 lux

EFFECT PARAMETERS MEASURED
- Determination of frond number: yes on days 0, 3, 5 and 7
- Determination of biomass: dry weight
- Determination of frond area: no
- Other: frond size, appearance, root length and number of colonies present, calculation of growth rate and yield

RANGE-FINDING STUDY
- Test concentrations: 0, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes (effect on growth (15-29%) at 10 and 100 mg/L)

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Any visual signs of phytotoxicity (abnormalities): no all fronds appeared green (except on day 5 at 100 mg/L some staining was observed)
- Decrease in frond size: none observed
- root size: slightly decreased at 10, 32 and 100 mg/L
- doubling time: 1.83 days

Results with reference substance (positive control):

Separate study with dichlorophenol at 0.625, 1.25, 2.5, 5.0 and 10 mg/L gave EC50 values between 3.0 and 3.4 for growth rate and 1.4 and 1.8 mg/L for frond number and dry weight

Reported statistics and error estimates:

Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981), and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955)

Inhibition of Average Specific Growth Rate and Yield Based on Frond number

Nominal Concentration (mg/L)	Average Specific Growth Rate		Yield
	0 – 7 Day	% Inhibition	0 – 7 Day	% Inhibition
0	0.319±0.016	-	84±10	-
1.0	0.319±0.011	0	83±8	1
3.2	0.306±0.014	4	75±8	11
10	0.294±0.003	8	69±5	18
32	0.281±0.015	12	62±7	26
100	0.256±0.012	20	50±5	40

 

Inhibition of Average Specific Growth Rate and Yield Based on dry weight

Nominal Concentration (mg/L)	Average Specific Growth Rate		Yield
	0 – 7 Day	% Inhibition	0 – 7 Day	% Inhibition
0	0.380±0.018	-	8.7 ± 1.2	-
1.0	0.390± 0.008	[3]	9.4 ± 0.6	[8]
3.2	0.354± 0.021	7	7.2 ± 1.2	17
10	0.331± 0.006	13	6.0 ± 0.3	31
32	0.324± 0.020	15	5.7 ± 0.8	34
100	0.296± 0.010	22	4.5 ± 0.4	48

 

Validity criteria fulfilled:

yes

Conclusions:

The EC50 for growth rate for the substance is > 100 mg/L
All EC50 values derived were > 100 mg/L. The NOEC for growth rate based on dry weight is 3.2 mg/L

Executive summary:

Lemna minor was exposed to an aqueous solution of the substance at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for a period of 7 days, under constant illumination at a temperature of  24 ± 1 °C.  The number of fronds in each control and treatment group was recorded on days 0, 3, 5 and 7 along with observations on plant development. Measured concentrations were 71 -103% of nominal. Effects on yield and growth rate were observed at 32 mg/L based on frond count and at 10 mg/L based on dry weight. The EC50 is > 100 mg/L and the NOEC is 3.2 mg/L (worst case on dry weight).

Description of key information

Lemna minor was exposed to an aqueous solution of the substance at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for a period of 7 days, under constant illumination at a temperature of  24 ± 1 °C.  The number of fronds in each control and treatment group was recorded on days 0, 3, 5 and 7 along with observations on plant development. Measured concentrations were 71 -103% of nominal. Effects on yield and growth rate were observed at 32 mg/L based on frond count and at 10 mg/L based on dry weight. The EC50 is > 100 mg/L and the NOEC is 3.2 mg/L (worst case on dry weight).

Key value for chemical safety assessment

EC50 for freshwater plants:

100 mg/L

EC10 or NOEC for freshwater plants:

3.2 mg/L