Reaction mass of 5,12-dihydroquino[2,3-b]acridine-7,14-dione and aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate) and dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

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:

18 Apr 2017 to 29 Apr 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 5,12-dihydroquino[2,3-b]acridine-7,14-dione and aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate) and dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

EC Number:

909-092-5

Molecular formula:

Unspecified

IUPAC Name:

Reaction mass of 5,12-dihydroquino[2,3-b]acridine-7,14-dione and aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate) and dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

Test material form:

solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
The sample of the test item was stored at ambient temperature without exposure to light in a tightly sealed container.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

In the definitive test, total organic carbon (TOC) was analyzed since the test substance is a multiconstituent-substance. From each test item concentration, three abiotic samples (i.e. samples taken from separate test vessels which did not contain Lemna gibba) were collected at exposure initiation (each in a volume of 100 mL). From the control nine abiotic samples (i.e. samples taken from separate test vessels which did not contain Lemna gibba) were collected at exposure initiation.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTIONS
Appropriate amounts of the test item were weighed separately in glass flasks with 20X AAP medium and mixed with appropriate volume of the 20X AAP medium. The mixtures were visually heterogeneous (with visible undissolved particles). The mixtures and the control (20X AAP medium) were left for mechanical shaking for 48 hours (90 revolutions per minute, darkness). After 48 hours of mechanical shaking, the mixtures were heterogeneous. All mixtures and the control were filtrated, first through a conditioned paper filter (qualitative cellulose filter) and next through a conditioned nitrocellulose membrane filter (Millipore 0.22 Ym). Each filtrate was visually homogeneous (without any visible undissolved particles) clear and colourless.

Test organisms

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: Duckweed
- Strain: CPCC 310
- Source: Canadian Phycological Culture Centre (CPCC), Department of Biology, University of Waterloo, Ontario, Canada.
- Method of cultivation: Cultivated in a standard laboratory culture at the Institute of Industrial Organic Chemistry, Branch Pszczyna, Department of Ecotoxicology, Laboratory of Aquatic Toxicology, according to the OECD Guideline No. 221 (2006) recommendations.

CULTURING
Duckweed Lemna gibba was transferred from agar bevels to the fresh 20X AAP medium in glass beakers with a capacity of 600 mL with transparent lids and incubated at room temperature with constant illumination (pre-culturing). The duckweed culture was inoculated to fresh medium once a week. A pre-culture was started eight days before exposure. Only organisms in good physiological condition without any discolouration were used for the inoculation of the test item concentrations and the control.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Hardness:

316 mg/L as CaCO3

Test temperature:

24.4 - 24.8 °C

pH:

- Fresh samples: 7.44 - 7.69
- Spent samples: 8.19 - 8.98

Conductivity:

1582 µS/cm at study initiation

Nominal and measured concentrations:

- Nominal loading rates: 0 (control), 6.4, 16, 40, 100, 250 and 625 mg/L (based on a range-finding study)
- Measured TOC concentrations in fresh samples: - Measured TOC concentrations in samples after 24 hours: See 'Any other information on materials and methods incl. tables'

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes, a thermostatic chamber
- Test vessel: Glass crystallisers with a depth of 4 cm and a diameter of 7 cm were used.
- Type: Closed; transparent lids were used to minimise evaporation and accidental contamination, allowing necessary air exchange.
- Fill volume: 150 mL
- Renewal rate of test solution: Daily
- No. of colonies per vessel: 3
- No. of fronds per colony: 3
- No. of vessels per concentration: 3
- No. of vessels per control: 6
- Other: The test vessels were conditioned with test item concentrations/control before exposure initiation and at renewals.

GROWTH MEDIUM
- Standard medium used: The 20X AAP medium recommended by the OECD Guideline No. 221 (2006) was the culturing medium for test organism and the diluent/solvent for the test item.

TEST MEDIUM / WATER PARAMETERS
- Alkalinity: 294 mg/L as NaHCO3 in the culture medium
- Culture medium different from test medium: No
- Intervals of water quality measurement: Daily, before and after renewals. Temperature was measured continuously.

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination
- Light intensity and quality: 7530 - 7740 lux
- Type of light: fluorescent (cool white light)

EFFECT PARAMETERS MEASURED
Number of fronds distinctly visible in each test vessel was counted and recorded as well as changes in plant development on days 2, 4 and after 7 days of exposure. At the same time, observations of plant development were performed: frond size, shape and appearance (necrosis, chlorosis, gibbosity or bending of fronds), colony break-up or loss of buoyancy, root length and appearance. The dry weight of the representative sample of the duckweed culture used as the inoculum was measured at after exposure initiation. The dry weight of all plants from each test vessel was measured after exposure termination. All colonies (with roots) were transferred onto previously weighed microscopic slides and dried at approximately 60ºC in a laboratory oven until constant weight.

PRELIMINARY NON-GLP TEST
- Test item loading rates: 0 (control), 0.1 (diluted), 1 (diluted), 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: see 'Any other information on materials and methods incl. tables'. Inhibition of growth rate and yield (based on frond number) was observed from loading rates of 10 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

> 625 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

7 d

Dose descriptor:

EL10

Effect conc.:

430.54 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: 302.16 - 692.59 mg/L

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

> 625 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield based on frond number

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EL10

Effect conc.:

30.26 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 15.40 - 47.44

Details on results:

During exposure, in all test item concentrations no distinctive changes from the development of plants in the control were observed: The test solutions were present as clear colourless solutions between renewals. In 'Any other information on results incl. tables' the results are tabulated and additional effect values are presented.

Results with reference substance (positive control):

The test was performed between 24 Mar 2017 and 31 Mar 2017. Relevant effect levels based on frond number:
- 7-d ErC50: 16.117 mg/L (95% CI: 14.871 – 17.696 mg/L)
- 7-d ErC10: 9.378 mg/L (95% CI: 8.482 – 10.200 mg/L)
- 7-d EyC50: 12.250 mg/L (n.d.)
- 7-d EyC10: 7.087 mg/L (n.d.)

Reported statistics and error estimates:

Probit method calculations and analysis by Shapiro-Wilk’s Test on Normal Distribution, Levene’s Test on Variance Homogeneity (with Residuals), Williams Multiple Sequential t-test Procedure. For the calculations and statistical analyzes, the ToxRat Professional commercial software was used.

Any other information on results incl. tables

Table: Inhibition of growth rate and yield

Test item concentration	Based on frond number		Based on dry weight
	% Inhibition at exposure termination (day 7) (growth rate)	% Inhibition at exposure termination (day 7) (yield)	% Inhibition at exposure termination (day 7) (growth rate)	% Inhibition at exposure termination (day 7) (yield)
Control	0	0	0	0
Filtrate of a loading of 6.4 mg/L	0.5	1.4	0.5	1.7
Filtrate of a loading of 16 mg/L	3.7	10.1	4.5	14.7
Filtrate of a loading of 40 mg/L	5	13.4	5.4	17.2
Filtrate of a loading of 100 mg/L	5.7	15.3	6.2	19.6
Filtrate of a loading of 250 mg/L	7.5	19.4	10.2	30.2
Filtrate of a loading of 625 mg/L	11.7	28.7	15.2	41.7

Table: section-by-section growth rate, growth rate and yield based on frond number

Test item concentration	Section-by-section growth rate			Growth rate	Yield**
	0-2 d	2-4 d	4-7 d	0-7 d	0-2 d	0-4 d	0-7 d
Control mv	428	433	319	383	122	413	1220
Filtrate of a loading of 6.4 mg/L mv	431	429	315	381	123	413	1203
Filtrate of a loading of 16 mg/L mv	431	405	302	368	123	390	1097
Filtrate of a loading of 40 mg/L mv	431	395	297	363	123	380	1057
Filtrate of a loading of 100 mg/L mv	424	399	293	361	120	377	1033
Filtrate of a loading of 625 mg/L mv	407	389	257	338	113	353	870

** yield = (frond number on day 7) – (frond number on day 0)

mv = mean value

EFFECT VALUES

7-d ErL20 based on frond number: > 625 mg/L

7-d LOErLR based on frond number: 16 mg/L

7-d NOErLR based on frond number: 6.4 mg/L*

*The statistical tests performed with data on growth rate based on frond number were: Shapiro-Wilk’s Test on Normal Distribution confirmed normal distribution of data, Levene’s Test on Variance Homogeneity showed that variances are homogeneous, Williams Multiple Sequential t-test Procedure showed a significant difference between test item loading rates in the range of 16 – 625 mg/L compared with the control.

7-d EyL20 based on frond number: 205.09 mg/L (95% CI: 149.48 - 286.84 mg/L)

7-d LOEyLR based on frond number: 16 mg/L

7-d NOEyLR based on frond number: 6.4 mg/L*

*The statistical tests performed with data on yield based on frond number were: Shapiro-Wilk’s Test on normal distribution confirmed normal distribution of data, Levene’s Test on variance homogeneity showed that variances are homogeneous, Williams Multiple Sequential t-test Procedure showed a significant difference between test item loading rates in the range of 16 – 625 mg/L compared with the control.

7-d ErL50 based on dry weight: > 625 mg/L

7-d ErL20 based on dry weight: > 625 mg/L

7-d ErL10 based on dry weight: 218.15 mg/L (95% CI: 173.10 – 273.27 mg/L)

7-d LOErLR based on dry weight: 16 mg/L

7-d NOErLR based on dry weight: 6.4 mg/L

7-d EyL50 based on dry weight: > 625 mg/L

7-d EyL20 based on dry weight: 72.56 mg/L( 95% CI: 53.77 – 92.88 mg/L)

7-d EyL10 based on dry weight: 15.02 mg/L (95% CI: 8.50 – 22.75 mg/L)

7-d LOEyLR based on dry weight: 16 mg/L

7-d NOEyLR based on dry weight: 6.4 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on materials and methods incl. tables'

Conclusions:

The test substance is with high probability not acutely harmful to aquatic plants.