Trisodium [μ-[3-[[4'-[[6-benzamido-1-hydroxy-3-sulpho-2-naphthyl]azo]-3,3'-dihydroxy[1,1'-biphenyl]-4-yl]azo]-4,5-dihydroxynaphthalene-2,7-disulphonato(7-)]]dicuprate(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:

experimental study

Adequacy of study:

key study

Study period:

2020

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 11127/E TE
Batch: 2024 (Thailand)
Purity: 63.1% total inorganics, 36.9% total organics
Physical State/Appearance: Blue solid
Expiry Date: 06 February 2024
Storage Conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Samples of the freshly prepared test media on Days 0 and 2, and of the old media on Day 5 were taken for chemical analysis in order to determine the concentration of copper present.
An additional sample of each loading rate was prepared on Day 0 and incubated alongside the test until Day 7. Duplicate samples were taken on each occasion and stored frozen for further analysis if required.

Vehicle:

yes

Details on test solutions:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2019 and Singer et al 2000), is to expose organisms to a WAF of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: Lemna
- Strain: minor
- Source: A culture of Lemna minor was obtained from Canadian Phycological Culture Centre, University of Waterloo, Ontario, Canada.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

24 ±1 °C

pH:

6.9 to 7.6

Nominal and measured concentrations:

Definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flask
- Type: closed
- Material, size, headspace, fill volume: Size 500 mL
- Aeration: No
- Agitation: No
- No. of colonies per vessel: Each control and test flask was inoculated with 3 colonies of Lemna minor (total 10 fronds).
- No. of vessels per concentration (replicates): Two
- No. of vessels per control (replicates): Two

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the prepared culture medium was adjusted, if necessary, to 6.5 ±0.2 with either 1M HCl or NaOH
- Photoperiod: continuous illumination for 7 days.
- Light intensity and quality: 7000 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: Manual counting
- Determination of biomass: dry weight

RANGE-FINDING STUDY
- Test concentrations: nominal loading rates of 1.0, 10 and 100 mg/L.
Results used to determine the conditions for the definitive study: The results showed no significant effect on growth at the test concentration of 1.0 mg/L
loading rate WAF. However, growth was observed to be reduced at 10 and 100 mg/L loading rate WAF. Based on this information nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

96 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

97 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

yield

Key result

Duration:

7 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOELR

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

yield

Results with reference substance (positive control):

Positive control 3,5-dichlorophenol test results;
EC50 Growth rate Frond number: 3.4 mg/L.
EC50 Growth rate Dry weightr: 2.7 mg/L.
EC50 Yeild Frond number: 2.4 mg/L.
EC50 Yeild Dry weight: 2.3 mg/L.

NOEC Growth rate Frond number: 1.25 mg/L.
NOEC Growth rate Dry weightr: 1.25 mg/L.
NOEC Yeild Frond number: 0.625 mg/L.
NOEC Yeild Dry weight: 1.25 mg/L.

Reported statistics and error estimates:

One way analysis of variance incorporating 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), were carried out on the average specific growth
rate and yield data at 7 days for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999-2001).

Validation of Mixing Period:

Preliminary investigational work indicated that there was no significant increase in the amount of copper obtained when the preparation period was extended for longer than 24 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

Range finding test:

Verification of Test Concentrations: Chemical analysis of the freshly prepared test media on Day 0 showed measured copper concentrations to range from 0.013 to 2.5 mg/L. Analysis of the freshly prepared test media on Day 2 showed measured copper concentrations to range from 0.012 to 2.3 mg/L. Analysis of the corresponding aged preparations on Day 5 showed measured copper concentrations to range from 0.0086 to 2.3 mg/L. Analysis of samples prepared on Day 0 and incubated alongside the test prior to analysis on Day 7 showed measured copper concentrations to range from 0.0083 to 2.0 mg/L. Given the slight decline in the concentration of copper obtained between periods of media renewal it was considered appropriate to conduct the definitive test using a semi-static testing regime to maximize the concentrations of copper to which the lemna were exposed.

Definitive Test

Verification of test concentrations: Chemical analysis of the freshly prepared loading rates on Days 0, 2 and 5 showed measured copper concentrations to range from 0.015 to 2.6 mg/L (equivalent to 0.49 to 86 mg/L as test item based on a test item copper content of 2.99%). Chemical analysis of the corresponding aged test preparations on Days 2, 5 and 7 showed measured copper concentrations to range from 0.013 to 2.7 mg/L (equivalent to 0.42 to 91 mg/L as test item). However, given that the toxicity cannot be attributed to a single component or a mixture of

components but to the test item as a whole, the results were based on nominal loading rates only.

Validation Criteria

The following data show that the doubling time of the control cultures was 1.61 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 : 10

Mean frond number in control cultures at day 7 : 162

Observations on Test Item Solubility

Observations on the test media were carried out during the mixing and testing of the WAFs. At the start and end of each mixing period, and following a 1-Hour settlement period the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF preparations were observed to have formed purple/blue dispersions, the intensity and depth of colour of which increased with increasing loading rate. Visual inspection of the WAFs at the end of settlement showed there to be dispersed test item present in all loading rate WAFs and as such it was considered appropriate to remove the aqueous phase for testing by filtration through two glass wool plugs and two sheets of coarse filter paper.

For each period of media renewal, microscopic observations of the 1.0, 3.2 and 10 mg/L loading rate WAFs following filtration showed there to be no micro-dispersions of test item present.

For the media renewal on Day 2, microscopic observations of the 32 mg/L loading rate WAF following filtration showed there to be no micro-dispersions of test item present. On Days 0 and 5 some particles of test item were observed to be present under microscopic observation.

For each period of media renewal, visual observations of the 100 mg/L loading rate WAF following filtration showed there to be dispersed test item present. It was considered that in instances where undissolved test item remained following filtration, that further filtration would not have removed anymore of the dispersed test item present.

At the start, and throughout the duration of the test all control cultures were observed to be clear colorless solutions. The test cultures were observed to range from being extremely slight pale purple/blue solutions at 1.0 mg/L loading rate WAF through to opaque purple/blue dispersions at 100 mg/L loading rate WAF.

Validity criteria fulfilled:

yes

Conclusions:

The EL50 of Average Specific Growth Rate (Frond number) in Lemna minor study was found to be greater than 100 mg/L. Corresponding NOELR was reported to be 10 mg/L.

Executive summary:

A 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)”.

Due to the low aqueous solubility and complex nature of the test item for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.

Following a preliminary range-finding test, Lemna minor was exposed to WAFs of the test item at nominal loading rates 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 test solutions were renewed on Days 2 and 5.

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.

The concentration of copper in the test preparations was determined in the freshly prepared test solutions on Days 0, 2 and 5, and in the corresponding aged test solutions on Days 2, 5 and 7 by copper analysis with inductively coupled plasma with mass spectrometric detection (ICP-MS) using an external standard.

Chemical analysis of the freshly prepared loading rates on Days 0, 2 and 5 showed measured copper concentrations to range from 0.015 to 2.6 mg/L (equivalent to 0.49 to 86 mg/L as test item based on a test item copper content of 2.99%). Chemical analysis of the corresponding aged test preparations on Days 2, 5 and 7 showed measured copper concentrations to range from 0.013 to 2.7 mg/L (equivalent to 0.42 to 91 mg/L as test item).

However, given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Lemna minor to the test item gave the following results:

Response Variable	Measurement Variable	EL50 (mg/L)	No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Average Specific Growth Rate	Frond Number	>100	10	32
	Dry Weight	>100	32	100
Yield	Frond Number	96	3.2	10
	Dry Weight	97	10	32

Description of key information

The EL50 in terms of Average Specific Growth Rate (Frond number) in Lemna minor study was found to be greater than 100 mg/L. Corresponding NOELR was reported to be 10 mg/L.

Key value for chemical safety assessment

EC50 for freshwater plants:

100 mg/L

EC10 or NOEC for freshwater plants:

10 mg/L

Additional information

A 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)”.

Due to the low aqueous solubility and complex nature of the test item for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item. Following a preliminary range-finding test, Lemna minor was exposed to WAFs of the test item at nominal loading rates 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 test solutions were renewed on Days 2 and 5. 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.

Exposure of Lemna minor to the test item gave the following results:

Response Variable	Measurement Variable	EL50 (mg/L)	No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Average Specific GrowthRate	Frond Number	>100	10	32
	Dry Weight	>100	32	100
Yield	Frond Number	96	3.2	10
	Dry Weight	97	10	32