Methyl N-ethyl-N-[4-[(5-nitro-2,1-benzisothiazol-3-yl)azo]phenyl]-β-alaninate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-06-26 to 2017-06-30, with the definitive exposure phase from 2017-06-27 to 2017-06-30

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

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 (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Methyl N-ethyl-N-[4-[(5-nitro-2,1-benzisothiazol-3-yl)azo]phenyl]-β-alaninate

EC Number:

257-779-7

EC Name:

Methyl N-ethyl-N-[4-[(5-nitro-2,1-benzisothiazol-3-yl)azo]phenyl]-β-alaninate

Cas Number:

52239-04-0

Molecular formula:

C19H19N5O4S

IUPAC Name:

methyl N-ethyl-N-[4-[(5-nitro-2,1-benzisothiazol-3-yl)azo]phenyl]-β-alaninate

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The test concentration of Disperse Blue 148 was analytically verified via HPLC-DAD in the saturated solution, the dilutions and the control at the start of the exposure (0 hours). At the start of the exposure (0 hours), sampling was carried out after preparation of the loading levels. As the measured test item concentration was below the LOD in the saturated solution, the test item concentrations at the other loading levels were considered to be also below LOD.
At the end of the exposure (72 hours), no analytical monitoring was carried out, because a measured test item concentration above LOD was not observed at the start of the exposure.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the Saturated solution
A saturated solution with a nominal loading of 100 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 ± 1 hour (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 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. During filtration, the filter will always be kept covered.
The solution was checked via laser beam (Tyndall effect), which was negative.

Test loading
All terms related to concentration level are given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations. The saturated solution and four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution.
The test item loadings were selected based on the results of a non-GLP preliminary range finding test.

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

Test organisms

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): 4 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:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Not specified

Test temperature:

min.: 22.0 max.: 23.0 mean value: 22.5

pH:

Nominal test item loading pH-values
[% of the saturated solution] Start; 0 hours End; 72 hours
100 8.11 8.05
50.0 8.07 8.16
25.0 8.09 8.46
12.5 8.05 8.94
6.25 8.06 9.00
Control 8.14 8.99

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Nominal: 6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution
Measured: < LOQ

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 6839 cells/mL
- Control end cells density: Mean 2581142 cells/mL
- No. of vessels per concentration (replicates): 3
- 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 0.064a2EDTA 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: 5577 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 preliminary range finding test at the saturated solution.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

28.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 25.9 - 31.5

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

63.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 60.9 - 66.9

Effect concentrations 5

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

14.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 12.5 - 16.5

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

29.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 28.4 - 31.4

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 saturated solution was visually clear throughout the exposure period.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA ALDRICH, 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 2017-04-18 to 2017-04-21. The reference item toxicity is in the valid range following test facility SOPS. For results, see Table 9.

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 0.559 0.773 ± 0.540
95% confidence interval 0.538 – 0.581
Yield inhibition
EyC50 0.320 0.406 ± 0.287
95% confidence interval 0.242 – 0.341
SD = Standard deviation

Reported statistics and error estimates:

EL-values and statistical analyses
EL-values of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEL, LOEL and statistical analyses
The NOEL and LOEL were determined by calculation of statistically significant differences of growth rates and yield.
The following statistical tests were conducted:

Shapiro-Wilk’s test on normal distribution was done with a significance level 0.01.
Levene’s test on variance homogeneity was done with a significance level 0.01.
Multiple sequentially-rejective Welsh-t-test after Bonferroni-holm was done with a significance level 0.05.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
•Excel, MICROSOFT CORPORATION
•SigmaPlot, SPSS INC.
•GraphPad Prism, GRAPHPAD SOFTWARE, INC.
•ToxRat Version 3.2.1, ToxRat Solutions GmbH

Any other information on results incl. tables

Cell Densities

Nominal test item loading		Replicate		Cell density [cells/mL]
[% of the saturated solution]		No.		0 hours		24 hours		48 hours		72 hours
100		1		6839		3553		< LOQcell density		< LOQcell density
		2		6839		4425		4080		5417
		3		6839		4018		< LOQcell density		3301
		Mean		6839		3999		n.a.		n.a.
50.0		1		6839		15158		47183		326861
		2		6839		15880		78848		489898
		3		6839		15336		69139		372940
		Mean		6839		15458		65057		396566
25.0		1		6839		26935		206931		1552048
		2		6839		29612		222532		1635849
		3		6839		23725		217008		1717601
		Mean		6839		26757		215490		1635166
12.5		1		6839		28945		312758		2409893
		2		6839		30619		367357		2555022
		3		6839		35013		357374		2432598
		Mean		6839		31526		345830		2465838
6.25		1		6839		33398		380243		2476488
		2		6839		34399		410194		2363779
		3		6839		36137		390882		2502881
		Mean		6839		34645		393773		2447716
Control		1		6839		32658		364724		2165689
		2		6839		40389		477960		2576733
		3		6839		40751		506178		2770551
		4		6839		28743		353114		2676978
		5		6839		40304		442064		2549638
		6		6839		31997		397717		2747260
		Mean		6839		35807		423626		2581142

 

LOQ_{cell density}  = Limit of quantification of the cell density (2736 cells/mL).             n.a.       = not applicable

 

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
[% of the saturated solution]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		n.a.	100		n.a.	100
	2		-0.0777	100		-1422	100
	3		-0.243	100		-3538	100
	Mean	(+)	n.a.	100	(-)	n.a.	100
50.0	1		1.29	35		320022	88
	2		1.42	28		483059	81
	3		1.33	33		366101	86
	Mean	(+)	1.35	32	(+)	389727	85
25.0	1		1.81	9		1545209	40
	2		1.83	8		1629010	37
	3		1.84	7		1710762	34
	Mean	(+)	1.83	8	(+)	1628327	37
12.5	1		1.96	1		2403054	7
	2		1.97	0		2548183	1
	3		1.96	1		2425759	6
	Mean	(-)	1.96	1	(-)	2458999	4
6.25	1		1.96	1		2469649	4
	2		1.95	1		2356940	8
	3		1.97	0		2496042	3
	Mean	(-)	1.96	1	(-)	2440877	5
Control	1		1.92			2158850
	2		1.98			2569894
	3		2.00			2763712
	4		1.99			2670139
	5		1.97			2542799
	6		2.00			2740421
	Mean		1.98			2574303

n.a. = not applicable

 

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	1.56	2.41	1.78	1.92	0.441	23.0	22.9
	2	1.78	2.47	1.69	1.98	0.430	21.7
	3	1.79	2.52	1.70	2.00	0.451	22.5
	4	1.44	2.51	2.03	1.99	0.537	27.0
	5	1.77	2.40	1.75	1.97	0.365	18.5
	6	1.54	2.52	1.93	2.00	0.492	24.6
				Mean	1.98
				SD ±	0.03
				CV [%]	1.53

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Disperse Blue 148 was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item loadings): The EL50-values with 95% confidence intervals for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were 63.7 (60.9 – 66.9) and 29.8 (28.4 – 31.4) % of the saturated solution, respectively. The NOEL-values for both inhibition of growth rate and yield after 72 hours were 12.5 % of the saturated solution, respectively. All effects are far above the water solubility limit of the test item.

Executive summary:

The toxicity of Disperse Blue 148 to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016 C.3 from 2017-06-26 to 2017-06-30, with the definitive exposure phase from 2017-06-27 to 2017-06-30 at the test facility. The aim of the study was the determination of NOEL, LOEL, EL₁₀, EL₂₀- and EL₅₀-values of growth rate and yield over a period of 72 hours.

 

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A saturated solution with a nominal loading of 100 mg test item/L was prepared with dilution water 24±1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out. The saturated solution was stirred for 24 ±1hour (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by membrane filtration. The following filtrate, i.e. the saturated solution was used in the test. The solution was checked via laser beam (Tyndall effect), which was negative.

The saturated solution and four dilution levels out of the saturated solution were tested in the definitive test in a geometrical series with a dilution factor of 2:6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution. Three replicates were tested for each test item concentration and six replicates for the control. The test media were colourless and clear throughout the test period. The environmental conditions were within the acceptable limits.

The measured concentrations of the test item were all below the limit of detection at the start of the exposure (0 hours), therefore no analysis at the end of the exposure was necessary.

 

All effect values given are based on the nominal test item loadings of DisperseBlue 148.

 

NOEL, LOEL, EL_x-values and 95 % Confidence Intervals ofDisperseBlue 148(0 - 72 hours)

              based on the nominal test item loadings[% saturated solution]

	Growth Rate Inhibition nominal test item loadings [% saturated solution]
NOEL	12.5
LOEL	25.0
ErL10	28.7 (25.9 – 31.5)
ErL20	39.8 (37.7 – 41.5)
ErL50	63.7 (60.9 – 66.9)
	Yield Inhibition nominal test item loadings [% saturated solution]
NOEL	12.5
LOEL	25.0
EyL10	14.6 (12.5 – 16.5)
EyL20	19.4 (17.9 - 20.9)
EyL50	29.8 (28.4 – 31.4)