Copper, [29H,31H-phthalocyaninato(2-)-.kappa.N29,.kappa.N30,.kappa.N31,.kappa.N32]-, sulfonated, sodium salts

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 07 October 2020 to 19 January 2021

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:

adopted 23 March 2006

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2 – 8°C), protected from light
- Stability during storage: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor.

Analytical monitoring:

yes

Details on sampling:

At the start of four of the daily media renewals (Day 0, 1, 4 and 6), 10 mL new media triplicate (A, B and C) samples were taken from the freshly prepared control and test media flask. At the end of the four of the daily media renewals (Day 1, 2, 5 and 7), 10 mL old media triplicate (A, B and C) samples were taken from pooled Lemna and individual no Lemna control and test vessels. The samples were taken using an air displacement pipette fitted with a plastic pipette tip. Samples were standardly sampled into 20 mL amber glass scintillation vials.

- Sample storage conditions before analysis:
Following collection of the samples, the “A”, “B” and “C” samples were transferred to the chemistry department under ambient conditions for storage and analysis, as appropriate. The Day 0 (new), 5 (old) and 7 (old) “A” samples were analysed on the day of sampling, all other “A” samples were frozen upon receipt and analysed after a maximum storage of 2 days. All “B” and “C” samples were frozen upon receipt.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: The 100 mg active ingredient/L solution was prepared by adding between 379.01 – 379.25 mg of test substance to 1000 mL of 20X AAP media.
- Controls: the highest concentration was checked for the Tyndall effect. All concentrations were checked by chemical analysis after day 1, 2, 5, and 7.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The turbidity meter confirmed that there was no dispersed test substance present.
- Other relevant information: As the test substance was light sensitive, the test substance was weighed and its solutions prepared under red light. The preparations were made in either amber glassware or glassware wrapped in foil to prevent light exposure.

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: Swollen duckweed
- Source: internal culture originally obtained from the University of Jena, Germany
- Method of cultivation:
Lemna gibba were cultured in approximately 300 mL of medium in glass beakers under continuous light (6500 to 10000 Lux). Initial monocultures consisted of approximately 50 - 100 fronds per vessel. During sub-culturing, an appropriate number of fronds were transferred into a clean beaker with new medium. Usually ca 50 fronds are sufficient, but more may have been sub-cultured if being prepared for use in a test. The actual number of fronds transferred did not need to be recorded.
In order to maintain healthy cultures, care was taken when selecting fronds for use in sub-culturing. Typically, young, rapidly growing plants light green in colour and visibly free of contamination by other organisms such as algae were chosen. Healthy plants of Lemna gibba may consist of colonies comprising up to seven fronds.

ACCLIMATION
- Period. 7 to 10 days before starting the test
- Medium: 20X AAP medium.
- Conditions: at 24 ± 2ºC in an incubator, or under an alternative light source (e.g. fluorescent light bank).

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

see table 1

Test temperature:

see table 1

pH:

see table 2

Conductivity:

see table 1

Nominal and measured concentrations:

Nominal: 0.32, 1, 3.2, 10, 32 and 100 mg AI/L, control;
Geometric mean measured: 0.109, 0.440, 1.99, 8.52, 30.3 and 98.4 mg AI/L, control

Details on test conditions:

TEST SYSTEM
- Test vessel: glass crystallising dishes containing ca 100 mL of the appropriate media (minimum 20 mm depth of media).
- Agitation: no
- Renewal rate of test solution: daily
- No. of fronds per vessel: 12
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
A no Lemna vessel was also prepared for analytical purposes.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis water
- Culture medium: 20X AAP culture medium (same as test medium)
- Intervals of water quality measurement: pH was measured daily (see table 2)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: adjusted to 7.5 ± 0.1 with either 0.1 M HCL or NaOH.
- Photoperiod: continious
- Light intensity and quality: monitored during test for 8 positions (see table 1). The samples were re-positioned randomly every day.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: manual counting
- Determination of biomass: dry weight
- Other: Plants were examined and any differences in general health in comparison to the control were noted. The occurrence of chlorosis or necrosis was recorded and other abnormalities such as changes in root length or colony break up were recorded, if applicable

RANGE-FINDING STUDY
- Test concentrations: Control, 1.25, 2.5, 5, 10 and 20 mg/L
- Results used to determine the conditions for the definitive study: EC50 (7 d) = 7.17 mg/L (frond numbers) and = 5.92 mg/L (dry weight)

Reference substance (positive control):

yes

Remarks:

Test was not part of the current GLP study (conducted in September 2020 under GLP regulations)

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

8.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

dry weight

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

85 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

dry weight

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

8.52 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

dry weight

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 98.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.109 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

frond number

Details on results:

Observations: see table 4

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC 50 (7d) = 5.92 mg/L based on dry weight

Reported statistics and error estimates:

Statistical analysis was performed using the CETIS program v 1.8.6.8. based on geometric mean measured concentrations.

DETERMINATION OF EFFECT CONCENTRATIONS
- A Williams Multiple Comparison test was used for the frond number Day 5 and 7 average specific growth rate and Day 7 yield calculations, because the data showed a monotonic and parametric trend.
- A Dunnett Multiple Comparison test was used for the frond numbers Day 3 average specific growth rate and the dry weight Day 7 average specific growth rate and yield calculations, because the data showed a non-monotonic but parametric trend.
- Linear interpolation analysis was performed in order to estimate EC10, EC20 and EC50 values for the Day 3, 5 and 7 frond number and Day 7 dry weight parameters
- Outlier analysis was performed by using Grubbs extreme value, no outliers were identified

Table 3: Results from definitive test; AI = Active ingredients

	Number of Fronds		Dry Weight (g)
	Day 0-7 Yield (mg AI/L)	Day 0–7 Average Specific Growth Rate (mg AI/L)	Day 0 - 7 Yield (mg AI/L)	Day 0 - 7 Growth Rate (mg AI/L)
EC10	0.499	2.82	0.169	8.64
EC20	1.41	14.7	0.383	22.0
EC50	16.8	>98.4	34.3	85.0
LOEC	0.440	0.440	0.440	30.3
NOEC	0.109	0.109	0.109	8.52

 

Table 4: Observations og Lemna fronds during test

Nominal Active Ingredient Concentration (mg/L)	Vessel Number	Observations
		Day 3	Day 5	Day 7
Control	1	100% Normal Fronds/Colonies with long roots	100% Normal Fronds/Colonies with long roots	100% Normal Fronds/Colonies with long roots
	2
	3
0.32	4	100% Normal Fronds/Colonies with long roots	100% Normal Fronds/Colonies with long roots	100% Normal Fronds/Colonies with long roots
	5
	6
1	7	100% Normal Fronds/Colonies with long roots	100% Normal Fronds/Colonies with long roots	100% Normal Fronds/Colonies with long roots
	8
	9	95% Normal Fronds/Colonies with long roots; 5% Chlorotic Fronds	90% Normal Fronds/Colonies with long roots; 10% Chlorotic Fronds	90% Normal Fronds/Colonies with long roots; 10% Chlorotic Fronds
3.2	10	90% Normal Fronds/Colonies with long roots; 10% Chlorotic Fronds	90% Normal Fronds/Colonies with long roots; 10% Chlorotic Fronds	90% Normal Fronds/Colonies with long roots; 10% Chlorotic Fronds
	11	100% Normal Fronds/Colonies with long roots	95% Normal Fronds/Colonies with long roots; 5% Chlorotic Fronds	95% Normal Fronds/Colonies with long roots; 5% Chlorotic Fronds
	12	95% Normal Fronds/Colonies with long roots; 5% Chlorotic Fronds
10	13	100% Normal Fronds/Colonies with long roots	95% Normal Fronds/Colonies with long roots (Lemna changed colour due to test substance); 5% Chlorotic Fronds	95% Normal Fronds/Colonies with long roots (Lemna changed colour due to test substance); 5% Chlorotic Fronds
	14		100% Normal Fronds/Colonies with long roots (Lemna changed colour due to test substance)	100% Normal Fronds/Colonies with long roots (Lemna changed colour due to test substance)
	15		95% Normal Fronds/Colonies with long roots (Lemna changed colour due to test substance); 5% Chlorotic Fronds	95% Normal Fronds/Colonies with long roots (Lemna changed colour due to test substance); 5% Chlorotic Fronds
32	16	100% Normal Fronds/Colonies with long roots	100% Normal Fronds/Colonies with long roots (Lemna changed colour due to test substance)	100% Normal Fronds/Colonies with long roots (Lemna changed colour due to test substance)
	17
	18
100	19	100% Normal Fronds/Colonies, 50% with long roots, 50% with short roots	100% small fronds (Lemna changed colour due to test substance)	100% small fronds (Lemna changed colour due to test substance)
	20
	21

Validity criteria fulfilled:

yes

Conclusions:

After 7 days the EC50 (dry weight, growth inhibition) for Lemna gibba was 85 mg/L.

Executive summary:

The effects of the test substance on the vegetative growth of the aquatic plant Lemna gibba were determined over a period of 7 days. The test was conducted in accordance with OECD Chemicals Testing Guideline No. 221 Lemna sp., Growth Inhibition Test (adopted 23 March 2006) using a semi-static regime with media renewals at daily intervals.
Based on geometric mean measured active ingredient concentrations, the Day 7 EyC50 and the Day 0-7 ErC50 values for frond numbers were calculated to be 16.8 and >98.4 mg/L, respectively. The corresponding NOEC values for yield and specific growth rate after 7 days was 0.109 mg/L.
Based on geometric mean measured active ingredient concentrations, the Day 7 EyC50 and the Day 0-7 ErC50 values for dry weight were calculated to be 34.3 and 85.0 mg/L, respectively. The corresponding NOEC values for yield and specific growth rate after 7 days were 0.109 and 8.52 mg/L, respectively.
All validity criteria for the OECD 221 guideline were met, therefore the test was considered valid.

Description of key information

Acutely harmful for aqautic organisms

The growth of lemna gibba was inhibited by the test item, based on the dry weight a EC 50 of 85 mg/L was observed.

Most likely no chronic effects on aquatic plants are expected.

 

Key value for chemical safety assessment

EC50 for freshwater plants:

85 mg/L

EC10 or NOEC for freshwater plants:

8.52 mg/L

Additional information

 

The effects of the test substance on the vegetative growth of the aquatic plant Lemna gibba were determined over a period of 7 days. The test was conducted in accordance with OECD Chemicals Testing Guideline No. 221 Lemna sp., Growth Inhibition Test (adopted 23 March 2006) using a semi-static regime with media renewals at daily intervals.

Based on geometric mean measured active ingredient concentrations, the Day 7 EyC50 and the Day 0-7 ErC50 values for frond numbers were calculated to be 16.8 and >98.4 mg/L, respectively. The corresponding NOEC values for yield and specific growth rate after 7 days was 0.109 mg/L.

Based on geometric mean measured active ingredient concentrations, the Day 7 EyC50 and the Day 0-7 ErC50 values for dry weight were calculated to be 34.3 and 85.0 mg/L, respectively. The corresponding NOEC values for yield and specific growth rate after 7 days were 0.109 and 8.52 mg/L, respectively.

All validity criteria for the OECD 221 guideline were met, therefore the test was considered valid.