Disodium [6-hydroxy-5-[(2-hydroxy-5-sulphophenyl)azo]naphthalene-2-sulphonato(4-)]cuprate(2-)

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:

toxicity to aquatic plants other than algae

Remarks:

Toxicity to the Aquatic Plant Lemna gibba

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD, EU guideline andGLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 761/2009, C.26.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium [6-hydroxy-5-[(2-hydroxy-5-sulphophenyl)azo]naphthalene-2-sulphonato(4-)]cuprate(2-)

EC Number:

276-423-1

EC Name:

Disodium [6-hydroxy-5-[(2-hydroxy-5-sulphophenyl)azo]naphthalene-2-sulphonato(4-)]cuprate(2-)

Cas Number:

72152-71-7

Molecular formula:

C16H8CuN2O8S2.2Na

IUPAC Name:

Cuprate(2-), [6-(hydroxy-kO)-5-[2-[2-(hydroxy-kO)-5-sulfophenyl]diazenyl-kN1]-2-naphthalenesulfonato(4-)]-, sodium

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples were taken at the end of the test from all test media and the control of the actual test by pouring together the contents of each treatment.
Additional samples of the control were taken at test start and test end.

Test solutions

Vehicle:

no

Details on test solutions:

Test Concentrations: 200, 50, 12.5, 3.125, 0.781, 0.195 and 0.049 mg test item/L and a control
corresponding to following mean measured concentrations of the test item:
200, 48.6, 12.0, 2.8, 0.5*, 0.25** and 0.25**mg test item/L
*For values < Limit of Quantification half the Limit of Quantification (1 mg test item/L) was used
**For values < Limit of Detection the Limit of Detection (0.25 mg test item/L) was used.
Control: In the control, test medium was used without addition of the test item.
Dosage of Test Item: The test medium of the highest test concentration of nominal 200 mg test item/L was prepared by dissolving 216.2 mg test item into 1081 mL test water by intense stirring for 10 minutes and ultrasonic treatment for 2 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
The test media were prepared just before introduction of the Lemna (= start of the test).

Appearance of the Test Item
in Test Medium:
The test media were coloured depending on the test item concentration from slightly rose to dark red .

Test organisms

Test organisms (species):

Lemna gibba

Details on test organisms:

Species: Lemna gibba G 3
Origin: The fronds introduced in the test were taken from ibacon's in-house laboratory culture.
Breeding Conditions: The plants are cultured in the laboratories of ibacon under standardised conditions according to the test guidelines.
Pre-Culture: For at 5 days under test conditions with weekly media exchange.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Test temperature:

22 to 24 °C

pH:

7.5 to 7.6 at test start; 8.4 to 8.5 at test end

Nominal and measured concentrations:

200, 50, 12.5, 3.125, 0.781, 0.195 and 0.049 mg test item/L and a control
corresponding to following mean measured concentrations of the test item:
200, 48.6, 12.0, 2.8, 0.5*, 0.25** and 0.25**mg test item/L
*For values < Limit of Quantification half the Limit of Quantification (1 mg test item/L) was used
**For values < Limit of Detection the Limit of Detection (0.25 mg test item/L) was used.

Details on test conditions:

Test Environment: Controlled environment room
Water Temperature: The temperature was measured daily in a test vessel filled with water and incubated under the same conditions as the test vessels.
22 to 24 °C
pH-Values: The pH-value of each treatment was measured at test start and at test end.
7.5 to 7.6 at test start
8.4 to 8.5 at test end
Light Regime: Continuous illumination
Light Intensity: 7747 Lux (mean value) with a range of 6600 to 8260 lux.
The illumination was achieved by fluorescent tubes, installed above the test vessels. Measurements were performed once during the test at 6 places distributed over the experimental area at the surface of the test media. Differences in light intensity over the test area did not exceed ± 15 %.

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the Lemna gibba and the experimental conditions the substance 3,5-dichlorophenol is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

14.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 2

Duration:

7 d

Dose descriptor:

EC50

Basis for effect:

growth rate

Remarks on result:

not determinable

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

601.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

13.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 6

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

0.36 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

4.19 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

4.28 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 9

Duration:

7 d

Dose descriptor:

EC10

Basis for effect:

frond number

Remarks on result:

not determinable

Effect concentrations 10

Duration:

7 d

Dose descriptor:

EC10

Remarks on result:

not determinable

Effect concentrations 11

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.27 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 12

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.323 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 13

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.25 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 14

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.25 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 15

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.25 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 16

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.25 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 17

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 18

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 19

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 20

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Growth Inhibition: The 7-day EyC50 was calculated to be 14.1 and 13.7 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50 frond number could not be calculated due to mathematical reasons. The 7-day ErC50 dry weight was calculated to 601.7 mg test item/L.
The 7-day EyC10 could not be calculated due to mathematical reasons.
The 7-day ErC10 was calculated to be 0.27 and 0.323 mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 0.25 and 0.50 mg test item/L for frond number and dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be 0.25 and 0.50 mg test item/L for frond number and dry weight, respectively.

Shape of Fronds: The shape of fronds and colonies after the test period of 7 days was not different to those in the control up to and including the nominal test concentration of 0.195 mg test item/L.
At the higher test item concentrations the fronds showed deviations from the control replicates after 7 days; i.e. gibbous growth, shorted roots and smaller fronds. .

Reported statistics and error estimates:

The ErC50 and the EyC50 (see Definitions), the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis.
For the determination of the 7-day LOEyC and NOEyC values significant differences at the test concentrations compared to the control values were tested by the Williams t-test (frond number and dry weight).
For the determination of the 7-day LOErC and NOErC values significant differences at the test concentrations compared to the control values were tested by the Williams t-test (frond number and dry weight).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Doubling Time of Frond Number in Control: 2.1 days, validity criterion was met.

Conclusions:

The influence of the test item on the growth of the freshwater plant Lemna gibba was assessed in a static concentration-response test.
The 7-day EyC50 was calculated to be 14.1 and 13.7 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50 for frond number could not be calculated (> 200 mg test item/L) The 7-day ErC50 for dry weight was calculated to be 601.7 mg test item/L.
The 7-day NOEyC and the LOEyC were determined to be 0.25 and 0.50 mg test item/L for frond number and dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be 0.25 and 0.50 mg test item/L for frond number and dry weight, respectively.
At the start of the test 100 % of the nominal test concentrations were found (average of the nominal concentrations of 200, 50, 12.5 and 3.125 mg test item/L). After 7 days test duration, 92 % of the nominal value was determined (average of the nominal concentrations of 200, 50, 12.5 and 3.125 mg test item/L). During the test the Lemna were exposed to a mean of 96 % of nominal. All reported results refer to mean measured concentrations.

Executive summary:

Toxicity to the Aquatic Plant Lemna gibba in a Static Growth Inhibition Test

Purpose: The purpose of this study was to determine the inhibitory effect of the test item on the growth of the freshwater aquatic plant Lemna gibba.

For this purpose, cultures of Lemna gibba were exposed in a static test to various concentrations under defined conditions. The inhibition of growth in relation to control cultures were determined over a test period of 7 days.

The test method of application and the test system are recommended by the test guidelines and Lemna gibba is the recommended test species.

The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

Guidelines/Recommendations:

Commission Regulation (EC) No 761/2009, Annex, Part C, C.26.: "Lemna sp. Growth Inhibition Test", Official Journal of the European Union (EN), dated August 24, 2009

OECD Guideline 221: "Lemna sp. Growth Inhibition Test", adopted March 23, 2006

SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

 

Material and Methods

Test Species: Lemna gibba G 3

Test Design: This study encompassed 8 treatment groups (7 dose rates of the test item and a control) with three replicates per test concentration and control.

At test start 12 fronds were introduced in each replicate and incubated for 7 days under static conditions. The frond numbers were determined on day 2, 5 and 7. The dry weight of each replicate was determined at test termination.

Endpoints: Yield and growth rate based on frond number and dry weight.

Test Concentrations: 200, 50, 12.5, 3.125, 0.781, 0.195 and 0.049 mg test item/L and a control

corresponding to following mean measured concentrations of the test item:

200, 48.6, 12.0, 2.8, 0.5*, 0.25** and 0.25**mg test item/L

*For values < Limit of Quantification half the Limit of Quantification (1 mg test item/L) was used

**For values < Limit of Detection the Limit of Detection (0.25 mg test item/L) was used.

Test Conditions: Water temperature: 22 to 24 °C; pH values at test start 7.5 to 7.6, at the end of the test 8.4 to 8.5; continuous illumination; mean light intensity: 7747 lux (6600 to 8260 lux).

Conclusion:

The influence of the test item on the growth of the freshwater plant Lemna gibba was assessed in a static concentration-response test.

The 7-day EyC50 was calculated to be 14.1 and 13.7 mg test item/L for frond number and dry weight, respectively.

The 7-day ErC50 for frond number could not be calculated (> 200 mg test item/L) The 7-day ErC50 for dry weight was calculated to be 601.7 mg test item/L.

The 7-day NOEyC and the LOEyC were determined to be 0.25 and 0.50 mg test item/L for frond number and dry weight, respectively.

The 7-day NOErC and the LOErC were determined to be 0.25 and 0.50 mg test item/L for frond number and dry weight, respectively.

At the start of the test 100 % of the nominal test concentrations were found (average of the nominal concentrations of 200, 50, 12.5 and 3.125 mg test item/L). After 7 days test duration, 92 % of the nominal value was determined (average of the nominal concentrations of 200, 50, 12.5 and 3.125 mg test item/L). During the test the Lemna were exposed to a mean of 96 % of nominal. All reported results refer to mean measured concentrations.

This study is classified acceptable and satisfies the guideline requirements for aquatic plant studies.