2,2'-[[4-[(2-bromo-6-chloro-4-nitrophenyl)azo]-3-chlorophenyl]imino]bisethanol

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 2018-12-10 to 2018-12-17, with the definitive exposure phase from 2018-12-11 to 2018-12-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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

Constituent 1

Reference substance name:

2,2'-[[4-[(2-bromo-6-chloro-4-nitrophenyl)azo]-3-chlorophenyl]imino]bisethanol

EC Number:

241-481-9

EC Name:

2,2'-[[4-[(2-bromo-6-chloro-4-nitrophenyl)azo]-3-chlorophenyl]imino]bisethanol

Cas Number:

17464-91-4

Molecular formula:

C16H15BrCl2N4O4

IUPAC Name:

2,2'-[[4-[(2-bromo-6-chloro-4-nitrophenyl)azo]-3-chlorophenyl]imino]bisethanol

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
Samples were analyzed with a HPLC-DAD method, implemented under non-GLP and documented finally in the GLP raw data.

Sampling schedule
All concentration levels and the control were analytically verified via HPLC-DAD at the start (0 hours) and at the end of the exposure (72 hours) with algae.

Sampling and pre-treatment
At the start, samples were taken from one additional replicate of the test item concentration and the control and at the end samples were taken from pooled replicates.

Criteria for the analytical monitoring (target)
Recoveries of the test item are not within ± 20% of the initially measured concentrations.

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 dilution water. The saturated solution was stirred for 24 ± 1 hour (1100 rpm, at room temperature) 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 was always kept covered.
The solution was checked via laser beam (Tyndall effect). The Tyndall effect 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 five dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of √10: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100% of the saturated solution. The test loadings are 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 item replicates.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK, CCAP 278/4
- Strain: CCAP 278/4
- Source (laboratory, culture collection):
Culture Collection of Algae and Protozoa (CCAP)
SAMS Research Services Ltd
Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK

- 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

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 7.91 7.70
31.6 7.90 7.69
10.0 7.89 7.90
3.16 7.92 8.06
1.00 7.93 8.12
0.316 7.95 8.40
Control 7.97 8.89

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 6442 cells/mL
- Control end cells density: Mean 1607801 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:

Ultrapure water was used to prepare the dilution water (conductivity max. 0.1 µS/cm)
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: 5873 lux,measured: 5064 - 6585 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 in 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.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

31.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

25.6

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 18.2 - 36.4

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 test media were clear and concentration related slightly orange coloured throughout the test period (possible turbidity related to algae growth not taken into account).
- 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 2018-09-25 to 2018-09-28. The reference item toxicity is in the valid range which was established by calculation of the average of the historic reference data since 2005, and the limits were set using the threefold standard deviation of these values.

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 0.799 0.753 ± 0.544
95% confidence interval 0.707 – 1.30
Yield inhibition
EyC50 0.607 0.407 ± 0.313
95% confidence interval 0.404 – 0.694
SD = Standard deviation

Reported statistics and error estimates:

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

NOEC, LOEC and analyses
The NOEC / LOEC was determined by calculation of statistical significance of growth rate and yield.
The following statistical tests were conducted:

Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01.
Levene’s test on variance homogeneity was done with a significance level of 0.01.
Monotonicity of Concentration/Response was calculated by Trend Analysis by Contrasts (significance level 0.05) for yield.
Monotonicity of Concentration/Response was calculated by non-parametric trend analysis by contrasts (significance level 0.05) for growth rate.
Williams Multiple Sequential t-test Procedure was performed with a significance level of 0.05 for yield.

Step-down Jonckheere-Terpstra test procedure was done with a significance level 0.05 for growth rate.

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 Professional, Version 3.3.0, TOXRAT SOLUTIONS GMBH

Any other information on results incl. tables

Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities. All effect values are given based on the nominal test item loadings.

 

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		6442		6303		20945		135244
		2		6442		5586		18264		127484
		3		6442		5744		17963		138298
		Mean		6442		5878		19057		133675
31.6		1		6442		11690		65796		537285
		2		6442		12013		68092		709322
		3		6442		14627		78569		713518
		Mean		6442		12777		70819		653375
10.0		1		6442		18770		167090		1468620
		2		6442		17323		129063		1443770
		3		6442		15498		104982		1219650
		Mean		6442		17197		133712		1377347
3.16		1		6442		15322		141247		1406085
		2		6442		17961		172770		1824287
		3		6442		26240		137285		1317286
		Mean		6442		19841		150434		1515886
1.00		1		6442		21742		190976		1523830
		2		6442		20903		158490		1644033
		3		6442		4108		172149		1172929
		Mean		6442		15584		173872		1446931
0.316		1		6442		27561		166901		1709422
		2		6442		24654		170083		1945582
		3		6442		22476		199620		1604139
		Mean		6442		24897		178868		1753048
Control		1		6442		24160		171573		1874484
		2		6442		25227		181026		1736804
		3		6442		23517		175024		1609427
		4		6442		15645		82536		1868997
		5		6442		24199		171101		1666698
		6		6442		17899		101333		929048
		Mean		6442		21775		147099		1614243

 

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		1.02	45		128802	92
	2		1.00	46		121042	92
	3		1.02	44		131856	92
	Mean	(+)	1.01	45	(+)	127233	92
31.6	1		1.48	20		530843	67
	2		1.57	14		702880	56
	3		1.57	14		707076	56
	Mean	(+)	1.54	16	(+)	646933	60
10.0	1		1.81	1		1462178	9
	2		1.80	2		1437328	11
	3		1.75	5		1213208	25
	Mean	(+)*	1.79	2	(-)	1370905	15
3.16	1		1.80	2		1399643	13
	2		1.88	-3		1817845	-13
	3		1.77	3		1310844	18
	Mean	(-)	1.82	1	(-)	1509444	6
1.00	1		1.82	1		1517388	6
	2		1.85	-1		1637591	-2
	3		1.74	5		1166487	27
	Mean	(-)	1.80	2	(-)	1440489	10
0.316	1		1.86	-2		1702980	-6
	2		1.90	-4		1939140	-21
	3		1.84	0		1597697	1
	Mean	(-)	1.87	-2	(-)	1746606	-9
Control	1		1.89			1868042
	2		1.87			1730362
	3		1.84			1602985
	4		1.89			1862555
	5		1.85			1660256
	6		1.66			922606
	Mean		1.83			1607801

* = statistically significant but biologically not relevant (< 5% inhibition)

 

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	1.32	1.96	2.39	1.89	0.538	28.4	33.6
	2	1.37	1.97	2.26	1.87	0.457	24.5
	3	1.30	2.01	2.22	1.84	0.484	26.3
	4	0.89	1.66	3.12	1.89	1.134	60.0
	5	1.32	1.96	2.28	1.85	0.485	26.2
	6	1.02	1.73	2.22	1.66	0.601	36.2
				Mean	1.83
				SD ±	0.09
				CV [%]	4.82

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Disperse Brown 4 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 > 100 and 25.6 (18.2 – 36.4)% of the saturated solution, respectively. The NOEL-values for both inhibition of growth rate and yield after 72 hours were 10.0% of the saturated solution, respectively. The effect levels are above the water solubility limit.

Executive summary:

The toxicity of Disperse Brown 4 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 2018-12-10 to 2018-12-17, with the definitive exposure phase from 2018-12-11 to 2018-12-14 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 6442 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, at room temperature) 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). The Tyndall effect was negative.

 

The saturated solution and five dilution levels out of the saturated solution were tested in the definitive test in a geometrical series with a dilution factor of√10:0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100% of the saturated solution. Three replicates were tested for each test item concentration and six replicates for the control. The test media were clear and concentration related slightly orange coloured throughout the test period. The environmental conditions were within the acceptable limits.

 

The concentrations of thetest item in all test concentrations and the control were analytically verified by HPLC-DAD at the start and at the end of the exposure.
The measured concentrations of the test item at the start of the exposure (0 hours) were < LOQ (0.14 mg/L) to 0.444 mg/L. The measured concentrations in the old media (72 h) were in the range of < LOQ to 82% of the initially values in all tested concentration levels.

 

All effect values given are based on the nominal test item loadings of DisperseBrown 4.

 NOEL, LOEL, EL_x-values and 95% Confidence Intervals of DisperseBrown 4 (0 - 72 hours)

 based on the nominal test item loadings

	Growth Rate Inhibition nominal test item loadings [% saturated solution]
NOEL	10.0
LOEL	31.6
ErL10	22.5 (16.5 – 26.9)
ErL20	37.6 (32.5 – 48.5)
ErL50	> 100
	Yield Inhibition nominal test item loadings [% saturated solution]
NOEL	10.0
LOEL	31.6
EyL10	7.16 (< 0.316 – 14.0)
EyL20	11.7 (6.60 – 18.1)
EyL50	25.6 (18.2 – 36.4)