16-nitroviolanthrene-5,10-dione

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

other: read across from similar substance

Adequacy of study:

key study

Study period:

2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

16,17-dimethoxyviolanthrene-5,10-dione

EC Number:

204-896-6

EC Name:

16,17-dimethoxyviolanthrene-5,10-dione

Cas Number:

128-58-5

Molecular formula:

C36H20O4

IUPAC Name:

16,17-dimethoxyanthra[9,1,2-cde]benzo[rst]pentaphene-5,10-dione

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the Saturated solution
A saturated solution with a nominal loading of 5 mg test item/L was prepared once 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out. The test item was applied onto a glass slide. The glass slide with the test item was inserted into a glass bottle with an appropriate amount of demineralized water. The saturated solution was stirred for 24 hours (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 10 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL was discarded. The following filtrate, i.e. the saturated solution, was used as a limit concentration in the test. During filtration, the filter was always kept covered.
The saturated solution was checked via laser beam (Tyndall effect). No Tyndall effect was observed. Then the components of the dilution water were added to the saturated solution.

Test loading
All terms related to concentration level will be given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations.
The limit loading of 5.00 mg/L was based on the results of a non-GLP preliminary range finding test. For results, see section 8.1.

Control
Six replicates (without test item) were exposed under the same conditions as the test item replicates.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Test temperature:

min.: 21.5 max.: 22.5 mean value: 22.0

pH:

Nominal Test Item Loading pH-value
[mg/L] Start; 0 hours End; 72 hours
5.00 8.10 8.46
Control 8.03 8.90

Salinity:

Not measured, freshwater conditions

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 6305 cells/mL
- Control end cells density: Mean 1319377 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 5212 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the range finding test at the saturated solution with a nominal loading of 5.00 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: EL10/20/50

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: EL10/20

Effect conc.:

< 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: The test item concentrations were clear during the exposure. The algae in the saturated solution were agglutinated after 72 hours. No agglutination of the algae was observed in the control replicates.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2016-10-11 to 2016-10-14 (with headspace). The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.435 0.764 ± 0.513
95 % confidence interval 0.414 – 0.458
Yield inhibition
EyC50 0.230 0.402 ± 0.293
95 % confidence interval 0.179 – 0.322
SD = Standard deviation

Reported statistics and error estimates:

EL-values and statistical analyses
EL-values of the growth rate and yield inhibition were estimated empirically based on the results of the single treatment level (Limit test design).

NOEL, LOEL and statistical analyses
NOEL/LOEL were determined by calculation of statistical significance of growth rates and yield. As a standard, a t-test was used for NOEL/LOEL calculations. When running a t-test, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

Software
All data were computer-generated and rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures.
Calculations and graphics were carried out using software
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.

Any other information on results incl. tables

Cell Densities

 

Nominal Test Item Loading	Replicate	Cell Density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours*	72 hours
5.00	1	6305	30148	231233	1251125
	2	6305	26887	185241	1014808
	3	6305	22331	162624	841834
	4	6305	28389	203939	1135870
	5	6305	22793	171025	951098
	6	6305	25291	172665	937765
	Mean	6305	25973	187788	1022083
Control	1	6305	34069	319837	1354892
	2	6305	24282	221688	1366422
	3	6305	25900	194936	1247855
	4	6305	28798	218714	1478533
	5	6305	27727	218821	1241258
	6	6305	20642	224713	1227304
	Mean	6305	26903	233118	1319377

     * = Direct measurement without dilution. Partially outside the internal calibration of the device, with no impact on integrity or validity of the data.

 

Evaluation after 72 hours

             Statistically significant differences of growth rates and yield compared to
                  control values are marked (+), not significant differences are marked (-).

 

Nominal Test Item Loading		Replicate		Growth Rate			Inhibition of Growth Rate		Yield			Inhibition of Yield
[mg/L]		No.		[d^-1]			[%]		[cells/mL]			[%]
5.00		1				1.76		1				1244820		5
		2				1.69		5				1008503		23
		3				1.63		8				835529		36
		4				1.73		3				1129565		14
		5				1.67		6				944793		28
		6				1.67		6				931460		29
		Mean		(+)		1.69		5		(+)		1015778		23
Control		1				1.79						1348587
		2				1.79						1360117
		3				1.76						1241550
		4				1.82						1472228
		5				1.76						1234953
		6				1.76						1220999
		Mean				1.78						1313072

 

negative inhibition = increase of growth

 

 

Section-by-Section and Average Specific Growth Ratesof the Control Group  (0 – 72 hours)

	Replicate No.	Specific Growth Rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.69	2.24	1.44	1.79	0.408	22.8	21.7
	2	1.35	2.21	1.82	1.79	0.432	24.1
	3	1.41	2.02	1.86	1.76	0.313	17.8
	4	1.52	2.03	1.91	1.82	0.266	14.6
	5	1.48	2.07	1.74	1.76	0.293	16.7
	6	1.19	2.39	1.70	1.76	0.603	34.3
				Mean	1.78
				SD ±	0.02
				CV [%]	1.37

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The nominal 72-h ErL10 and ErL50 were > 5.00 mg/L.
The tested substance has no inhibitory effect on freshwater green algae.

Executive summary:

The toxicity of the tested substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 Method C.3. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

A saturated solution of 5.00 mg test item/L was prepared one day prior to the start of the exposure.The study was conducted under static conditions with an initial cell density of 6305 cells/mL. Based on a preliminary range finding test, the saturated solution was tested in a limit test. Six replicates were used for the limit loading level and the control. The environmental conditions were within the acceptable limits.

 The test media were visually clear throughout the test period. The algae in the saturated solution were agglutinated after 72 hours. No agglutination of the algae was observed in the control replicates. No analytical monitoring was carried out. Due to the low solubility, a specific analytical method was not available and the concentrations are too low for unspecific analysis as the determination of the total organic carbon content. Therefore, all effect values given are based on the nominal test item concentration.