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

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate)

EC Number:

278-207-2

EC Name:

Aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate)

Cas Number:

75431-69-5

Molecular formula:

C20H12N2O5S.1/3Al

IUPAC Name:

aluminum tris(7,14-dioxo-5,7,12,14-tetrahydroquino[2,3-b]acridine-2-sulfonate)

Constituent 2

Reference substance name:

5,12-dihydroquino[2,3-b]acridine-7,14-dione

EC Number:

213-879-2

EC Name:

5,12-dihydroquino[2,3-b]acridine-7,14-dione

Cas Number:

1047-16-1

Molecular formula:

C20H12N2O2

IUPAC Name:

5,12-dihydroquino[2,3-b]acridine-7,14-dione

Constituent 3

Reference substance name:

Dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

EC Number:

243-319-2

EC Name:

Dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

Cas Number:

19795-24-5

Molecular formula:

C20H12N2O8S2.2/3Al

IUPAC Name:

dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

From each test item concentration, three abiotic 100 mL samples (taken from separate vessels which did not contain Lemna gibba) were collected at exposure initiation. One of them was transferred for chemical analyses at exposure initiation, whereas the other were stored und test concitions for 24 h (first renewal) and the other were stored under test conditions till exposure temrination and transferred for chemical analysis.
For the control nine 100 mL abiotic samples were collected. Three samples respectively were analysed in analogy to the test item concentrations.

Test solutions

Vehicle:

no

Details on test solutions:

Test Concentrations
filtrates of loadings of 625, 250, 100, 40, 16 and 6.4 mg/L plus the control.
Control
In the control, test medium 20X AAP was used without addition of the test item.
Preparation of test item solutions
appropriate amounts of the test item were weighed separately in glass flasks with 20X AAP medium and mixed with the appropriate volume 20X AAP. The mixtures and the control were left for mechanical shaking for 48 hours at 90 rpm in the dark. The heterogeneous mixture was subsequently filtered through a membrane filter (0.22 µm, Millipore) yielding visually clear filtrates.

Test Concentrations of the preliminary non-GLP study
filtrates of loadings of 100 and 10 mg/L; 10fold and 100fold diluted filtrate of the 10 mg/L loading.

Test organisms

Test organisms (species):

Lemna gibba

Details on test organisms:

Species: Lemna gibba CPCC 310
Origin: The culture on agar bevels was received from Canadian Phycological Culture Centre (CPCC), Department of Biology, University of Waterloo, Ontario, Canada
Culturing media: 20X AAP as recommended by OECD 221 is used as culturing medium and diluent/solvent for the test item.
Method of cultivation: The plants are cultured in the laboratories of Institute of Industrial Organic Chemistry, Branch Pszczyna under standardised conditions according to the test guidelines. Duckweed was transferred from agar bevels to the fresh 20X AAP medium in glass beakers with a capacitiy of 600 mL with transparent lids and incubated at room temperature with a constant illumination (pre-culturing). The 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 discoloration were used for the inoculation of the test item concentrations and the control.

The sensitivity of the duckweed culture is monitored with the reference material 3,5-dichlorophenol.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Hardness:

316 mg/L CaCO3

Test temperature:

24.4 to 24.8 °C

pH:

7.44 - 7.69 (fresh test item concentrations and control)
8.19 - 8.98 (spent test item concentrations and control)

Nominal and measured concentrations:

nominal: 625, 250,100, 40, 16 and 6.4 mg /L and a control.

Details on test conditions:

The test item concentrations were split up into three replicates, the control was split up into six replicates. The test was performed in glass crystallizers (depth 4cm, diameter 7 cm) containing 150 mL of each treatment per replicate. Transparent lids were used to minimize evaporation and accidental contamination, allowing necessary air exchange. During exposure the test vessels were arranged at random and daily repositioned.
The test was performed in a semi-static design with daily renewals.
At exposure initiation three colonies consisting of three fronds of similar size were chosen and transferred with inoculating loop from the preculture into each test vessel. Each replicate was inoculated with a total of 9 fronds. The total number of fronds in each test vessel was counted on days 2, 4 and at exposure termination. At the same time, observations of plant development wer performed: frond size, sahpe and appearance (e.g. necrosis, chlorosis), colony break-up or loss of buoyancy, root length.
The dry weight of the representative sample of the duckweed culture used as the inoculum was measured at exposure initiation and after exposure termination.

Light Regime: Continuous illumination
Light Intensity: 7530 - 7740 Lux (mean value)
Incubation chamber used: constant conditions throughout the test is provided by thermostatic chamber
Temperature: the temperature is continously recorded by electronic device with asensor submerged in an additional test vessel with 150 mL of test medium

RANGE-FINDING STUDY
- Test concentrations: in a non-GLP preliminary study the following concentrations were tested: nominal 100 mg/L and 10 mg/L; 10fold and 100fold diluted filtrate of a loading of 10 mg/L
- Results used to determine the conditions for the definitive study: the stability of the test substance could not be demonstrated with the unspecific analytical method. Therefore worst case exposure conditions with daily renewal of the test medium was applied to keep the test conditions as stable as possible.
Based on the results the concentrations for the main study were defined.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 625 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 2

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 625 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 625 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 625 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

205.09 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL

Effect concentrations 6

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

72.56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL

Effect concentrations 7

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

> 625 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 8

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

> 625 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 9

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

30.26 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL

Effect concentrations 10

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

15.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL

Effect concentrations 11

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

430.54 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL

Effect concentrations 12

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

218.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL

Effect concentrations 13

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

6.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 14

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

6.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 15

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

6.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 16

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

6.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 17

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 18

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 19

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 20

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

the morphology of plants (shape, color, colonies and fronds, appearance of roots) was observed in each test item concentration and the controls on days 2, 4 and at exposure termination. The morphological effects were compared with appearance of colonies in the control.
During exposure, in all test item concentraitons no distinctive changes from the development of the plants in the control were observed.

Results with reference substance (positive control):

the test with the reference material 3,5-dichlorophenol (Sigma-Aldrich, purity 97 %) was performed under semi-static conditions with two renewals during 7 day exposure. Five concentrations of the reference substance ranging from 0.32 to 3.2 mg/L wer used. Each concentration was split up into 3 replicates, whereas the conctrol into six replicates. The following endpoint values, based on nominal concentrations, were calculated:

frond number dry weight

ErC50 16.117 11.370
ErC20 11.294 9.005
ErC10 9.378 7.972
EyC50 12.250 9.085
EyC20 8.552 7.305
EyC10 7.087 6.518

The results of the test with the reference substance prove the validity of the test system and test conditions at the test facility.

Reported statistics and error estimates:

Probit method calculations and anylsis by Shapiro-Wilk's Test on Normal Distribution, Levene's Test on Variance Homogeneity (with Residuals), Williams Multiple Sequential t-test Procedure

The ErC50 and the EyC50 (see definitions), the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis.
For the determination of the 7-day LOEyC and NOEyC values significant differences at the test concentrations compared to the control values were tested by the Williams t-test (frond number and dry weight).
For the determination of the 7-day LOErC and NOErC values significant differences at the test concentrations compared to the control values were tested by the Williams t-test (frond number and dry weight).

Any other information on results incl. tables

Table 1: Growth rate and yield inhibition after exposure termination (day 7), preliminary test (non-GLP)

Concentration	Based on frond number		Based on dry weight
	% inhibition (growth rate)	% inhibition (yield)	% inhibition (growth rate)	% inhibition (yield)
Control	0.00	0.00	0.00	0.00
100fold diluted filtrate (10 mg/L loading)	0.00	0.00	0.00	2.23
10fold diluted filtrate (10 mg/L loading)	0.00	0.00	1.97	6.93
10 mg/L, filtered	3.27	10.70	3.70	12.32
100 mg/L, filtered	2.69	8.75	6.40	20.17

Table 2: Growth rate and yield inhibition after exposure termination (day 7), definite study

Nominal concentration of filtered solutions	Based on frond number		Based on dry weight
	% inhibition (growth rate)	% inhibition (yield)	% inhibition (growth rate)	% inhibition (yield)
Control	0.00	0.00	0.00	0.00
6.4 mg/L	0.5	1.4	0.5	1.7
16 mg/L	3.7	10.7	4.5	14.7
40 mg/L	5.0	13.4	5.4	17.2
100 mg/L	5.7	15.3	6.2	19.8
250 mg/L	7.5	19.4	10.2	30.2
625 mg/L	11.7	28.7	15.2	41.7

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The influence of the test item on the growth of the freshwater plant Lemna gibba was assessed in a semi-static concentration-response test.
The 7-day EyC50 was calculated to be > 625 mg/L for frond number and dry weight.
The 7-day ErC50 was calculated to be > 625 mg/L for frond number and dry weight.

Executive summary:

The toxicity of the test item to the aquatic plant Lemna gibba in a growth inhibition test was determined based on the procedures indicated by the following internationally accepted methods:

- OECD 221 equivalent to EU C. 26

Biological Results

Growth Inhibition:

The 7-day EyC50was calculated to be> 625 mg test item/L for frond number and dry weight. The 7-day ErC50was calculated to be> 625 mg test item/L for frond number and dry weight. The 7-day EyC10was calculated to be 30.26 and 15.02 mg test item/L for frond number and dry weight, respectively. The 7-day ErC10was calculated to be 430.54 and 218.15 mg test item/L for frond number and dry weight, respectively. The 7-day NOEyC and the LOEyC were determined to be 6.4 and 16 mg test item/L for frond number and dry weight, respectively. The 7-day NOErC and the LOErC were determined to be 10 and 32 mg test item/Lfor frond number and dry weight, respectively.

Shape of Fronds:

The shape of fronds and colonies after the test period of 7 days was not different to those in the control up to and including the nominal test concentration of 625 mg test item/L.

Analytical Results:

The quantification of the test item in the test samples was performed using TOC analysis. The TOC values at exposure initiation increased with the loading, but were not proportional. The values measured after 24 hours were in the range of 76 to 84 % of the initial value, indicating a slight decrease during the 24 hour renewal intervals. After 7 days under exposure conditions the values were generally approximately half of the inital values. But these values were of low relevance since the test solutions were renewed daily. The test item is a hardly soluble substance. Therefore, all reported results refer to nominal concentrations.