Benzenamine, N,N-dimethyl-, oxidized, molybdatetungstatephosphates

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Jan 2018 to 25 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[4-[bis[4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]-dimethyl-ammonium. molybdatetungstatephosphates

Molecular formula:

C25 H30 N3. P Moy Wz O40 (y +z = 12)

IUPAC Name:

[4-[bis[4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]-dimethyl-ammonium. molybdatetungstatephosphates

Test material form:

solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test substance was stored at ambient temperature.

Sampling and analysis

Analytical monitoring:

no

Remarks:

The test solutions were analysed for Molybdenum (Mo) concentration as a proxy for test item, as the test item cannot be quantified by analytical methods.

Test solutions

Vehicle:

no

Details on test solutions:

Test Item Preparation (especially for difficult test substances)
The test item is a difficult item as defined by the OECD Guideline 23 (2000) because it is a complex mixture with partial solubility, depending on loading rate. In order to maximise the amount of test item in solution, the test solutions were prepared as water accommodated fractions (WAF) following the OECD Guideline 23 (2000). Seven water accommodated fractions (WAF), one for each loading rate and one WAF control were prepared (Table 1) and allowed to stir for about 2 days. Stirring was followed by an approximately 4 hour settling period. WAF solutions were then removed from the WAF vessel and collected in a glass holding vessel by siphoning from the middle of the water column, discarding the first ~50 – 100 mL into an appropriate container. New sets of WAFs were prepared every 24 hours for a total of seven sets of WAFs to allow for daily test solution renewal.

Test organisms

Test organisms (species):

Lemna minor

Details on test organisms:

Test organism species: Lemna minor
Strain: CPCC 492
Source of test organism: Canadian Phycological Culture Centre, Waterloo, ON, Canada
Age of organism at test initiation: 10 days old
Method of cultivation: Test plants were transferred (sub-cultured) into sterile Swedish Standard (SIS) media 10 days prior to testing and held under the same conditions as used for the test. Test plants were selected from an established axenic stock culture which is maintained on a weekly basis by transferring to sterile growth medium (SIS or other nutrient rich media) and held under similar conditions as used for the test.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Test temperature:

24 - 25 °C

pH:

6.8-7.6

Nominal and measured concentrations:

The nominal test item loading rates tested were 0.1, 0.3, 0.9, 2.8, 8.3, and 25 mg Test Item/L along with a negative control (0) and a WAF control (0).

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass Erlenmeyer flasks resealed with a sterile foam plug
- Test volume: 125 mL
- No. of colonies per vessel: 9 fronds/ test vessel at the start of exposure
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Randomization: The test vessels were placed in the test chamber according to a randomization plan

GROWTH MEDIUM
- Standard medium used: yes; Swedish Standard Synthetic growth media (SIS)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Swedish Standard Synthetic growth media (SIS)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous 24-h light
- Light intensity and quality: Artificial light, 101 – 116 µE/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The frond number in the test vessels were counted at the start of the test, after day 3, day 5 and after end of exposure the total number of fronds was counted in each replicate. The appearance of the plants (e.g., colouration, chlorosis, necrosis, destroyed colonies, etc.) in each replicate was observed and recorded at test initiation, on Day 3 and Day 5, and at test completion. Observations on the appearance of the test solutions in the test vessels were also made and recorded at these time points.
The biomass based on dry weight was determined at the start of exposure from a sample of the inoculum culture representative of what was used to begin the test, and at the end of the test with the plant material from each treatment and control vessel. All colonies were collected from each of the test vessels and rinsed with deionized water. They were blotted to remove excess water and then dried at 60°C for a minimum of 24 hours to a constant weight. At test completion (day 7), the number of fronds in each test replicate were counted and recorded. Final visual observations on plant appearance and test solution appearance was conducted and recorded.

RANGE-FINDING STUDY
The test concentrations were selected on the basis of range finding tests (non-GLP, static renewal) using the nominal loading rates of 0, 0.1, 1.0, 10, and 100 mg Test Item/L. The estimated ELR50 for frond increase for this preliminary test was 2.2 mg Test Item/L (95% Confidence Limits: 1.5 mg/L, 3.2 mg/L), based on nominal loading rates. The corresponding estimated ELR50 for dry weight was 3.7 mg Test Item/L (95% Confidence Limits: 1.5 mg/L, 8.8 mg/L), based on nominal loading rates.A significant Tyndall effect was observed in the 100 mg/L loading rate, and a slight effect was observed in the 10 mg/L loading rate. This effect was not observed in the 0.1 mg/L or 1.0 mg/L loading rates. Due to this observation, an additional treatment was tested alongside this preliminary test; a portion of the 100 mg/L loading rate solution was centrifuged prior to testing to allow for comparison of any potential effects from particles in suspension. There was minimal difference in response between the centrifuged and non-centrifuged 100 mg/L loading rate treatments.

Reference substance (positive control):

yes

Remarks:

No reference item was employed in this study. However, a reference Item test using nickel sulphate hexahydrate (CAS 10101-97-0) was conducted as a separate test within one month of the study to assess the relative sensitivity of the test item.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

7.13 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

growth rate

Remarks on result:

other: Average specific growth rate based on frond number

Effect concentrations 2

Duration:

7 d

Dose descriptor:

EL10

Effect conc.:

< 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

growth rate

Remarks on result:

other: Average specific growth rate based on frond number

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

9.81 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

growth rate

Remarks on result:

other: Average specific growth rate based on dry weight

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EL10

Effect conc.:

< 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

growth rate

Remarks on result:

other: Average specific growth rate based on dry weight

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

0.69 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

biomass

Remarks on result:

other: Yield based on frond number

Effect concentrations 6

Duration:

7 d

Dose descriptor:

EL10

Effect conc.:

< 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

biomass

Remarks on result:

other: Yield based on frond number

Effect concentrations 7

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

0.87 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

biomass

Remarks on result:

other: Yield based on dry weight

Effect concentrations 8

Duration:

7 d

Dose descriptor:

EL10

Effect conc.:

< 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

biomass

Remarks on result:

other: Yield based on dry weight

Details on results:

Concentration control analysis was not performed since the test item is a complex mixture with partial solubility, depending on loading rate. In order to maximise the amount of test item in solution, the test solutions were prepared as water accommodated fractions (WAF). However, all reasonable efforts were taken to produce a WAF of the test substance in test media, following the guidance in OECD 23. The test organisms were exposed to a WAF of the test substance at the given loading rate and the effect concentrations should be expressed based on the loading rate as recommended in OECD 23. As the test item cannot be quantified by analytical methods, the test solutions were analysed for Molybdenum (Mo) concentration as a proxy for test item (Table 2). Analysis of aqueous samples involved direct analysis following any required dilution with 2% nitric acid. Analysis of the samples was performed by Inductively Coupled Plasma Mass Spectrometry (ICPMS).

Results with reference substance (positive control):

Reference Item Test Results
The 7-day EC25and 95% confidence limits based on frond increase for the reference item test are summarised in Table 3. This value was within the acceptable range (±2 SD)of previous tests conducted in this laboratory and indicates that the test organisms responded normally to the reference item.

Any other information on results incl. tables

Results on observations

At test completion, plants in the negative control and the WAF control appeared light green, healthy, with some transparent fronds. Plants in the 0.1 mg/L loading rate appeared light green, healthy, with some chlorosis. Plants in the 0.3 mg/L loading rate appeared light green, healthy, with some chlorosis and also shortened roots. Plants in the 0.9 mg/L appeared light green with chlorosis and shortened roots. Plants in the 2.8 mg/L loading rate appeared light green with chlorosis, some small fronds and some transparent fronds, as well as destroyed roots. Plants in the 8.3 mg/L loading rate appeared light green with chlorosis, some small fronds and some transparent fronds, as well as destroyed roots which appeared purple. Plants in the 25 mg/L loading rate appeared generally light green with chlorosis and loss of buoyancy, some small fronds, some transparent fronds, and some purple fronds, as well as destroyed roots which appeared purple.

Table 3: Summary of Lemna minor Reference Item Test

Test Initiation Date	7-day EC25 (mg Ni/L)	95% Confidence Limits (mg/L)	±2SD of Previous Mean (mg/L)	Calculation Method
2018 Jan 26	10.1	5.4, 16.5	4.0, 42.6	3P Log-Logistic

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test substance is acutely toxic for aquatic plants.