7,16-dichloro-6,15-dihydroanthrazine-5,9,14,18-tetrone

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-10-09 to 2016-10-13

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Test item
Vat Blue 6

Preparation of the Saturated solution
A saturated solution with a nominal loading of 5 mg test item/L was prepared once 24 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.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Not specified

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 8.23 8.45
Control 8.03 8.90

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

No analytical monitoring

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)

Duration:

72 h

Dose descriptor:

other: ErL10/20/50

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

other: EyL10/20

Effect conc.:

< 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

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.

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	28051	165511	871405
	2	6305	26633	154477	859425
	3	6305	26207	213814	1059038
	4	6305	28068	152563	696092
	5	6305	31521	173627	778237
	6	6305	22012	125766	655865
	Mean	6305	27082	164293	820010
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				1.64		8				865100		34
		2				1.64		8				853120		35
		3				1.71		4				1052733		20
		4				1.57		12				689787		47
		5				1.61		10				771932		41
		6				1.55		13				649560		51
		Mean		(+)		1.62		9		(+)		813705		38
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

 

 

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

Validity criteria fulfilled:

yes

Conclusions:

In this study a slight inhibition of growth on the freshwater green alga Pseudokirchneriella subcapitata was found after 72 hours exposure to the test item Vat Blue 6. The nominal EL50-values for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were > 5.00 mg/L, respectively. The nominal ErL10-values for inhibition of growth rate after 72 hours was > 5.00 mg/L, the ErL10 for yield < 5.00 mg/L, respectively.

Executive summary:

The toxicity of Vat Blue 6 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 from 2016-10-09 to 2016-10-13, with the definitive exposure phase from 2016-10-10 to 2016-10-13 at the test facility. 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 of Vat Blue 6.

 

EL-values of Vat Blue 6 (0 - 72 hours)

                  based on nominal test item loading [mg/L]

	Inhibition of Growth Rate nominal test item loading [mg/L]
ErL10	> 5.00
ErL20	> 5.00
ErL50	> 5.00
	Inhibition of Yield nominal test item loading [mg/L]
EyL10	< 5.00
EyL20	< 5.00
EyL50	> 5.00

Description of key information

In the algal study (OECD 201) a slight inhibition of growth on the freshwater green alga Pseudokirchneriella subcapitata was found after 72 hours exposure to the test item Vat Blue 6. The nominal EL50-values for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were > 5.00 mg/L, respectively. The nominal ErL10-values for inhibition of growth rate after 72 hours was > 5.00 mg/L and <5.00 mg/L for yield, respectively.