Reaction products of copper phthalocyanine, sulfuric acid, and sodium carbonate

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 August 05 to 31, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13

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:

A nominal amount of test item (200 mg) was dissolved in culture medium and the volume adjusted to 2 liters to give the required test concentration of 100 mg/L and mixed to ensure adequate mixing and homegeneity.The concentration and stability of the test item in the test preparations were verified by chemical analysis on Day 0 and Day 7.

Vehicle:

yes

Details on test solutions:

Range Finding Test
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 10 and 1.0 mg/L. The test was conducted in glass conical flasks (500 mL). Two replicate flasks each containing 250 mL were prepared for each control and test concentration. The test item was dissolved directly in culture medium. A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 10 and 1.0 mg/L.The control test item was culture medium alone. Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
A nominal amount of test item (200 mg) was dissolved in culture medium and the volume adjusted to 2 liters to give the required test concentration of 100 mg/L. The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.The control test item was culture medium alone.

Test organisms (species):

Lemna minor

Details on test organisms:

A culture of Lemna minor was obtained from Canadian Phycological Culture Centre, University of Waterloo, Ontario, Canada. Cultures were maintained in the laboratory by the periodic replenishment of culture medium . The culture was maintained in the laboratory at a temperature of 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) for at least 7 days prior to the start of the test.

Test type:

static

Water media type:

other: Culture medium in reverse osmosis purified water (Elga Optima 15+ or Elga Purlab Option R-15 BP)

Limit test:

yes

Total exposure duration:

7 d

Remarks on exposure duration:

Lemna minor was exposed to an aqueous solution of the test item at a nominal concentration of 100 mg/L (six replicate flasks for a period of 7 days, under constant illumination at a temperature of 24 ± 1 ºC.

Post exposure observation period:

None

Test temperature:

Temperature was maintained at 24 ± 1 ºC throughout the test.

pH:

Control : Day 0: 7.6 Day 7: 8.4 - 8.6100 mg/LDay 0: 6.9 - 7.0Day 7: 7.9 - 8.3

Nominal and measured concentrations:

100 mg/l nominal concentration

Details on test conditions:

Range Finding Test
The range-finding test was conducted by exposing Lemna minor to a series of nominal test concentrations of 1.0, 10 and 100 mg/L for a period of 7 days. At the start of the range-finding test the number of fronds present in each test and control culture was recorded along with observations on frond size, appearance, root length and number of colonies present. The flasks were then incubated at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) on a black non-reflective surface for 7 days. On Days 3 and 5 the test solutions were renewed, and observations on the test organisms were recorded on days 0, 3, 5 and 7. A static testing regime was employed.

Definitive Test
Each control and test flask was inoculated with 3 colonies of Lemna minor (total 9 fronds). The flasks were then incubated at 24 ± 1 ºC under constant illumination (intensity approximately 7000 lux) on a black non-reflective surface for 7 days.
A static testing regime was employed.

Reference substance (positive control):

yes

Remarks:

A positive control (Envigo Study Number MM01PC) used 3,5-dichlorophenol as the reference item at concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L.

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: frond numbers and dry weight

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: frond numbers and dry weight

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: frond numbers and dry weight

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: frond numbers and dry weight

Details on results:

Range-finding Test
The results showed no significant effect on growth at the test concentrations of 1.0, 10 and 100 mg/L. Based on this information a single test concentration of six replicates, of 100 mg/L was selected for the definitive test. This experimental design conforms to a “limit test” to confirm that at the maximum test concentration given in the OECD Test Guidelines, no effect on growth was observed. Chemical analysis of the 100 mg/L test preparations on Days 0 (fresh media) and Days 3 and 7 (old media) showed a measured concentration of 99 % of nominal was obtained indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the test preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to range from 98 % to 100 % of nominal and so the results are based on nominal test concentrations only.
Validation Criteria
The following data show that the doubling time of the control cultures was 1.81 days in line with the OECD Guideline that states the doubling time should be less than 2.5 days:
Mean frond number in control cultures at Day 0 : 9
Mean frond number in control cultures at Day 7 : 88

Growth Data Based on Frond Number
The following results are based on inhibition of average specific growth rate and yield were determined from the frond number data:
Average Specific Growth Rate
ErC10 (frond number) = >100 mg/L
ErC20 (frond number) = >100 mg/L
ErC50 (frond number) = >100 mg/L
Where:
ErCx = the test concentration that reduced average specific growth rate by x %.
Statistical analysis of the average specific growth rate data was carried out for the control and 100 mg/L test concentration using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences between the control and 100 mg/L test concentration (P 0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of average specific growth rates calculated from frond numbers was 100 mg/L.
Yield
EyC10 (frond number) = >100 mg/L
EyC20 (frond number) = >100 mg/L
EyC50 (frond number) = >100 mg/L
Where:EyCx = the test concentration that reduced yield by x %.
Statistical analysis of the yield data was carried out for the control and 100 mg/L test concentration. There were no statistically significant differences between the control and 100 mg/L test concentration (P ≥ 0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from frond numbers was 100 mg/L.

Growth Data Based on Dry Weight
The following results based on inhibition of average specific growth rate and yield were determined from the dry weight data:
Average Specific Growth Rate
ErC10 (dry weight) = >100 mg/L
ErC20 (dry weight) = >100 mg/L
ErC50 (dry weight) = >100 mg/L
Where:
ErCx = the test concentration that reduced average specific growth rate by x %.
Statistical analysis of the average specific growth rate data was carried out for the control and 100 mg/L test concentration. There were no statistically significant differences between the control and 100 mg/L test concentration (P 0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of average specific growth rate calculated from dry weight was 100 mg/L.
Yield
EyC10 (dry weight) = >100 mg/L
EyC20 (dry weight) = >100 mg/L
EyC50 (dry weight) = >100 mg/L
Where:
EyCx = the test concentration that reduced yield by x %.
Statistical analysis of the yield data was carried out for the control and 100 mg/L test concentration. There were no statistically significant differences between the control and 100 mg/L test concentration (P 0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from dry weight was 100 mg/L.

Observations
All test and control cultures were inspected on Days 0, 2, 5 and 7.

Water Quality Criteria
Temperature was maintained at 24 ± 1 ºC throughout the test.

Results with reference substance (positive control):

The results from the positive control with 3,5-dichlorophenol were within the normal ranges for this reference item.

Reported statistics and error estimates:

Average Specific Growth Rate based on frond numbers
Statistical analysis of the average specific growth rate data was carried out for the control and 100 mg/L test concentration using a Student’s t-test incorporating Bartlett's test for homogeneity of variance. There were no statistically significant differences between the control and 100 mg/L test concentration (P≥0.05) and therefore the NOEC in terms of inhibition of average specific growth rates calculated from frond numbers was 100 mg/L.
Yield based on frond numbers
Statistical analysis of the yield data was carried out for the control and 100 mg/L test concentration. There were no statistically significant differences between the control and 100 mg/L test concentration (P≥0.05) and therefore the NOEC in terms of inhibition of yield calculated from frond numbers was 100 mg/L.

Average specific growth rate based on dry weight
Statistical analysis of the average specific growth rate data was carried out for the control and 100 mg/L test concentration. There were no statistically significant differences between the control and 100 mg/L test concentration (P≥0.05) and therefore the NOEC in terms of inhibition of average specific growth rate calculated from dry weight was 100 mg/L.
Yield based on dry weight
Statistical analysis of the yield data was carried out for the control and 100 mg/L test concentration. There were no statistically significant differences between the control and 100 mg/L test concentration (P≥0.05) and therefore the "No Observed Effect NOEC in terms of inhibition of yield calculated from dry weight was 100 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

ECr50 > 100 mg/l
NOEC = 100 mg/l

Executive summary:

Method

The study was performed to assess the effect of the test item on the growth of the freshwater plant Lemna minor. The method followed that described in the OECD Guideline No. 221 “Lemna sp.Growth Inhibition Test (March 2006)”.

Following a preliminary range-finding test, Lemna minor was exposed to an aqueous solution of the test item at a nominal concentration of 100 mg/L (six replicate flasks 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, 2, 5 and 7 along with observations on plant development.

Results

Chemical analysis of the test preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to range from 98 % to 100 % of nominal and so the results are based on nominal test concentrations only.

The resulting ECr50 was more than 100 mg/l and the NOECr was 100 mg/l.

Description of key information

ECr50 > 100 mg/l

NOEC = 100 mg/l

Additional information

There is no experimental available data of the target substance, thus the information on the Similar Substance 01 was taken into account. The structural differences occurring between the target substance and Similar Substance 01 are not expected to significantly impact the toxicity to aquatic plants, thus the read across approach can be considered as representavive and appropriate (details in the document attached to the IUCLID section 13).

The study was performed to assess the effect of the Similar substance 01 on the growth of the freshwater plant Lemna minor. The method followed that described in the OECD Guideline No. 221 “Lemna sp.Growth Inhibition Test (March 2006)”.

Following a preliminary range-finding test, Lemna minor was exposed to an aqueous solution of the test item at a nominal concentration of 100 mg/l (six replicate flasks 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, 2, 5 and 7 along with observations on plant development.

Chemical analysis of the test preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to range from 98 % to 100 % of nominal and so the results are based on nominal test concentrations only.

The resulting ECr50 was more than 100 mg/l and the NOECr was 100 mg/l.