Trisodium [3-[[4-[[6-anilino-1-hydroxy-4-sulpho-2-naphthyl]azo]-5-hydroxy-o-tolyl]azo]naphthalene-1,5-disulphonato(5-)]cuprate(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:

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

Adequacy of study:

key study

Study period:

From February 27 to July 13, 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The complete read across justification is detailed in section 13. Source study has reliability 1.

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 0.032, 0.16, 0.8, 4.0, 20, 100 and 500 mg/l
- Samples of each test item concentration and control were collected at exposure initiation and at exposure termination.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The highest test item concentration was prepared by mixing an appropriate amount of the test item with the 20X AAP medium to obtain a test item concentration of 500 mg/L. The test item concentrations of 500 mg/L were visually homogeneous (without any visible undissolved particles), coloured and non-transparent. Lower test item concentrations was prepared by a series of dilutions with the test medium.
- Controls: 20X AAP medium

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Strain: Lemna gibba CPCC 310
- Source: cultivation in a standard laboratory culture at the Institute of Industrial Organic Chemistry Branch Pszczyna, Department of Ecotoxicology, Laboratory of Aquatic Toxicology. The culture of Lemna gibba on agar bevels was received from Canadian Physiological Culture Centre, Department of Biology, University of Waterloo, Ontario, Canada.
- Age of inoculum (at test initiation): the dry weight of the representative sample of the duckweed culture used as the inoculum.
- Method of cultivation: Duckweed Lemna gibba was transferred from agar bevels to the fresh 20X AAP medium in glass beakers with a capacity of 600 mL with transparent lids and incubated in 22 - 26 °C with constant illumination (pre-culturing). The duckweed culture was inoculated to fresh medium once a week.

ACCLIMATION
- Acclimation period: 7 days
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

341 mg CaCO3/l

Test temperature:

23.8 – 24.6 °C

pH:

at exposure initiation: 7.39 – 7.86
at exposure termination: 8.17 – 9.12

Nominal and measured concentrations:

nominal concentrations: 0, 0.032, 0.16, 0.8, 4.0, 20, 100, and 500 mg/l
measured concentrations: < LOD (= control), < LOD, < LOQ, 0.70, 3.37, 19.10, 84.36, 460.23 mg/l (at exposure initiation); < LOD (=control), < LOD, < LOQ, 0.70, 3.26, 18.12, 101.12, 450.23 mg/l (at exposure termination)

According to guideline the separating factor should not exceeded 3.2 but a larger value may be used when the concentration response curve is flat. Thus, a separation factor of 5.0 was used.

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes
- Test vessel: beakers
- Material, fill volume: glass, 400 ml
- No. of colonies per vessel: 3
- No. of fronds per colony: 3
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes (20X AAP medium)

TEST MEDIUM / WATER PARAMETERS
- According to guideline (20X AAP medium)

OTHER TEST CONDITIONS
- Light intensity and quality: 8770 – 9043 lux

EFFECT PARAMETERS MEASURED:
- Determination of frond number: on days 2, 4 and at exposure termination
- Determination of biomass: dry weight at exposure termination

RANGE-FINDING STUDY
- Test concentrations: 0, 1.0, 10 and 100 mg/l
- Results used to determine the conditions for the definitive study: The range of the test item concentrations to be used in the definitive test was determined on the basis of the results of the preliminary test. In addition, in the preliminary test in the modified 20X AAP medium (control) changes in morphology were observed when compared to the normal development of colonies in the 20X AAP medium. Therefore, in the definitive test the 20X AAP medium was used as the test medium.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

66.414 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate (based on frond number)

Remarks on result:

other: 95% CL: 29.835 – 193.167

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

0.556 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate (based on frond number)

Remarks on result:

other: 95% CL: 0.109 – 1.549

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.046 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate (based on frond number)

Remarks on result:

other: 95% CL: 0.004 – 0.201

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

1.407 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield (based on frond number)

Remarks on result:

other: 95% CL: 0.447 – 3.993

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

0.043 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield (based on frond number)

Remarks on result:

other: 95% CL: 0.003 – 0.170

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

< 0.032 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield (based on frond number)

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

42.863 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate (based on dry weight)

Remarks on result:

other: 95% CL: 22.347 – 93.041

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

1.048 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate (based on dry weight)

Remarks on result:

other: 95% CL: 0.283 – 2.462

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.151 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate (based on dry weight)

Remarks on result:

other: 95% CL: 0.022 – 0.491

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

1.341 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield (based on dry weight)

Remarks on result:

other: 95% CL: 0.579 – 3.017

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

0.097 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield (based on dry weight)

Remarks on result:

other: 95% CL: 0.015 – 0.262

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

< 0.032 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield (based on dry weight)

Details on results:

- Any visual signs of phytotoxicity (abnormalities): shorter roots at 20, 100 and 500 mg/l (on day 2, 4 and at exposure termination ); bent down fronds at 4 mg/l (at exposure termination), at 20 mg/l (on day 4 and at exposure termination) and at 100 mg/l (on day 2, 4 and at exposure termination); break up of colonies at 500 mg/l (on day 2 and 4)
- Necrosis / chlorosis: at 100 and 500 mg/l on day 4 and at exposure termination

Results with reference substance (positive control):

- Relevant effect levels: ErC50 = 11.76 mg/l, ErC20 = 8.09 mg/l, ErC10 = 6.65 mg/l, EyC50 = 8.91 mg/l, EyC20 = 7.18 mg/l, EyC10 = 6.41 mg/l based on frond number (at exposure termination)

Conclusions:

After 7 days of exposure, as for frond number:
- growth rate: ErC50 = 66.414 mg/l
- yield: EyC50 = 1.407 mg/l

After 7 days of exposure, as for dry weight:
- growth rate: ErC50 = 42.863 mg/l
- yield: EyC50 = 1.341 mg/l

Executive summary:

Method

According to OECD Guideline 221: 7 days exposure of Lemna gibba under static conditions. Effect parameters were frond number and dry weight; both of them were observed in terms of growth rate and yield.

Seven test item concentrations of 0.032, 0.16, 0.8, 4.0, 20, 100 and 500 mg/l plus the control were used in the test. The concentrations of the test item were determined with a validated spectrophotometric method.

Results

Samples collected at exposure initiation were in the range of 84.3 – 95.5 % of the nominal concentration, thus confirming correct preparation of the test item concentrations; samples collected at exposure termination were in the range of 81.4 – 101. 1% of the nominal concentration, thus showing that test item concentrations were stable under test conditions.

Since measured concentrations were within ± 20 % of nominal concentrations at the beginning and and the end of the study period, effect levels were expressed as nominal concentrations.

At exposure termination, in test item concentrations of 0.032, 0.16 and 0.8 mg/l no distinctive changes from the normal development of plants in the control were observed.

In test item concentration of 4.0 mg/l fronds were slightly bent down.

In the test item concentration of 20, 100 and 500 mg/l, shorter roots were observed. In test item concentrations of 100 and 500 mg/l spots of chlorosis or necrosis on fronds were observed. Moreover, in test item concentrations of 20 and 100 mg/l bent down fronds were observed, in the test item concentration of 500 mg/l break up of colonies was observed.

As for frond number:

- growth rate: ErC50 = 66.414 mg/l

- yield: EyC50 = 1.407 mg/l

As for dry weight:

- growth rate: ErC50 = 42.863 mg/l

- yield: EyC50 = 1.341 mg/l

Description of key information

Test conducted according to OECD guideline 221 using Lemna gibba. The following key values were determined based on frond number after an exposure period of 7 days:

ErC50 = 66.414 mg/l and ErC10 = 0.046 mg/l.

Key value for chemical safety assessment

EC50 for freshwater plants:

66.414 mg/L

EC10 or NOEC for freshwater plants:

0.046 mg/L

Additional information

The growth of Lemna gibba exposed to the test item was investigated in a 7 -day static test to determine test item concentrations causing 10, 20 and 50 % inhibition of growth rate (r) and yield (y).

Seven test item concentrations of 0.032, 0.16, 0.8, 4.0, 20, 100 and 500 mg/l plus the control were used in the test. The concentrations of the test item resulted to be ± 20 % of nominal values throughout the test duration.

The endpoint values after 7 days, expressed as nominal concentrations, resulted to be:

Based on frond number:

ErC50/7d: is 66.414 mg/l (95 % confidence limits 29.835 – 193.167)

EyC50/7d: is 1.407 mg/l (95 % confidence limits 0.447 – 3.993)

Based on dry weight:

ErC50/7d: is 42.863 mg/l (95 % confidence limits 22.347 – 93.041)

EyC50/7d: is 1.341 mg/l (95 % confidence limits 0.579 – 3.017)

For the purpose of classification, the ErC50 value of 66.414 mg/l referred to frond number was taken into account.