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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-28 to 2019-01-31

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

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
All loading rates and the control were analytically verified via HPLC-DAD in the fresh media at the start of exposure (0 hours) and in the old media at the end of the exposure (48 hours). The samples were analysed with a HPLC-DAD method, implemented under non-GLP and documented finally in the GLP raw data. The method was validated according to SANCO 3029/99 rev.4 (2000).

Sampling for the analytical monitoring
At the start of the exposure (0 hours), samples were taken from the freshly prepared loading rates and the control for analysis.
At the end of the exposure (48 hours), samples for the analyses of the old media were taken directly from test vessels containing daphnids.

Criteria for the analytical monitoring (target)
Recoveries of the test item should be within ± 20% of the nominal or 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 with dilution water once 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out. The solution was stirred for 23.5 hours (1100 rpm, room temperature, dark) 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. The following filtrate, i.e. the saturated solution, was used in the test. During filtration, the filter was always kept covered. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. No Tyndall effect was observed in the saturated solution. The saturated solution was used for the preparation of further dilution levels by dilution with dilution water.

Test item loading rates
6 loading rates of the test item in a geometric series with a separation factor of 2, prepared by dilution of the saturated solution with dilution water, were tested as follows: 0.313 - 0.625 - 1.25 - 2.50 - 5.00 - 10.0% of the saturated solution
The loading rates of the test item were based on the results of a non-GLP preliminary range finding test.

Control
Dilution water without test item incubated under the same conditions as the test groups

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system: Daphnia magna STRAUS (clone 5), obtained from continuous laboratory cultures.

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guideline.

Origin Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Breeder Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, in an incubator, 16 hours illumination;
light intensity of max. 1500 lx

Culture medium Elendt M4, according to OECD 202, Annex 3 (2004), is used. The composition of the culture medium is presented in the table below.

 

Composition of the Culture Medium Elendt M4
according to OECD 202, Annex 3 (2004)

Component Concentration [mg/L]
CaCl2 x 2 H2O 294
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 x 5 H2O 7.47
NaNO3 0.274
KH2PO4 0.143
K2HPO4 0.184
Na2EDTA x 2 H2O 2.50
FeSO4 x 7 H2O 0.996
H3BO3 2.86
MnCl2 x 4 H2O 0.361
LiCl 0.306
SrCl2 x 6 H2O 0.152
RbCl 0.0710
NaBr 0.0160
Na2MoO4 x 2 H2O 0.0615
CuCl x 2 H2O 0.0168
ZnCl2 0.0130
CoCl2 x 6 H2O 0.0100
KI 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamin 0.0010
Biotin 0.00075
pH 8.2 to 0.8

Feeding of the culture stocks
The daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae were cultured at the test facility.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Dilution water
0 hours: Total hardness [mg CaCO3/L]: 278

Test temperature:

target: 18 - 22°C, constant within ± 1°C
measured: 19 - 20 °C

pH:

Water Quality Parameters in the fresh Media at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) per loading rate and control)

Nominal loading rate pH-value Dissolved
of the saturated O2 concentration
test item solution
[%] [mg/L]
10.0 7.46 8.17
5.00 7.43 8.34
2.50 7.42 8.50
1.25 7.41 8.39
0.625 7.45 8.50
0.313 7.50 8.32
Control 7.59 8.50


Water Quality Parameters in the old Media at the End of the Exposure (48 hours)
(measured in one appropriate replicate (containing daphnids) per loading rate and control)

Nominal loading rate pH-value Dissolved Replicate number
of the saturated O2 concentration
test item solution
[%] [mg/L]
10.0 6.78 8.08 4
5.00 6.72 8.01 4
2.50 6.69 8.23 3
1.25 6.67 8.21 4
0.625 6.61 8.23 4
0.313 6.53 8.21 4
Control 6.53 8.09 4


Water Quality Parameters of the Dilution Water at the Start of the Exposure (0 hours)

Dilution water pH-value Dissolved Temperature Conductivity Total hardness
dated: O2 concentration [mg/L] [°C] [µS/cm] [mg CaCO3/L]
2019-01-29 7.59 8.50 19.8 583 278

Dissolved oxygen:

see above

Salinity:

Not applicable, freshwater

Conductivity:

Dilution water day 0: 583 [µS/cm]

Nominal and measured concentrations:

The measured concentrations of the test item were all below the LOQ (0.14 mg/L of the test item) at the start of the exposure (0 hours) and at the end of the exposure period (48 hours).

Measured Concentrations and Percent of the Nominal Concentration of the Test Item Disperse Brown 4 during the Definitive Test
Sampling date Fresh media, 0 hours Old media, 48 hours
Disperse Brown 4
Loading rate of the Meas. conc. Meas. conc.
saturated solution
[%] [µg/L] [µg/L]
10.0 < LOQ < LOQ
5.00 < LOQ < LOQ
2.50 < LOQ < LOQ
1.25 < LOQ < LOQ
0.625 < LOQ < LOQ
0.313 < LOQ < LOQ
Control < LOQ < LOQ

Meas. conc. = measured concentration of the test item, mean value of two injections (control one injection)
LOQ = limit of quantification (0.140 µg test item/L)

Details on test conditions:

Test method The study was performed under static conditions.

Test duration 48 hours

Test vessels Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Test volume 20 mL

Dilution water Dilution water Same composition as the culture medium

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for each loading rate and the control.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. To achieve this juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period were used for the test. No first brood progeny was used for the test.

Acclimatization
Acclimatization of the daphnids was not necessary, because the composition of the dilution water was equivalent to the culture medium.

Application
20 g test solution per replicate was weighed into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water by a pipette.

Test temperature (target) 18 - 22 °C, constant within ± 1 °C

Illumination (target) Diffuse light, light intensity of max. 1500 lx

Photoperiod (target) 16/8 hours light/dark cycle

Feeding The daphnids were not fed during the study.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

other: ELR50

Effect conc.:

6.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: confidence limits: 5.89 - 7.94 mg/L

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

other: ELR50

Effect conc.:

5.26 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: confidence limits: 4.55 - 6.75 mg/L

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2019 01-23 to 2019-01-24. For results of the most recent of the monthly performed reference tests, see the table below.

EC50-Value (with Confidence Limits) of the Reference Item Potassium dichromate
based on nominal concentrations [mg/L], (0 - 24 hours)
Current Study EC50 Confidence limits Valid Range
2.06 mg/L 1.00 - 4.00 mg/L 0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5

The respective confidence limits were empirically derived from the observation data as follows: The highest concentration level without any effect (EC0) and the lowest concentration level causing 100% immobilization (EC100) were used as confidence limits.

Reported statistics and error estimates:

Methods of evaluation
The ELR100-values after 24 and 48 hours were empirically derived from the observation data (lowest concentration level resulting in 100% immobilization). The effect levels (ELR10 / 50 / 100) are given based on the nominal loading rates, since the measured test item concentrations were below the LOQ during the exposure period.

ELR-values and statistical analyses
The ELR10- and ELR50-values (after 24 and 48 hours of exposure) were calculated by sigmoidal dose-response regression with the software GraphPad Prism. The respective 95% confidence limits were calculated from the standard error and the t distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism.

Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

Immobilization Ratesafter 24 and 48 hours of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

Nominal loading rate of the saturated test item solution [%]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
10.0	100	60	80	100	85	100	80	100	100	95
5.00	0	20	0	60	20	20	60	20	80	45
2.50	0	0	0	0	0	0	0	20	0	5
1.25	0	0	0	0	0	0	0	0	0	0
0.625	0	0	0	0	0	0	0	0	0	0
0.313	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Absolute Numbers of immobile daphnids after 24 and 48 hours of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

Nominal loading rate of the saturated test item solution [%]	Number of immobile Daphnids / Total number of Daphnids
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	Sum	1	2	3	4	Sum
10.0	5 / 5	3 / 5	4 / 5	5 / 5	17 / 20	5 / 5	4 / 5	5 / 5	5 / 5	19 / 20
5.00	0 / 5	1 / 5	0 / 5	3 / 5	4 / 20	1 / 5	3 / 5	1 / 5	4 / 5	9 / 20
2.50	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	1 / 5	0 / 5	1 / 20
1.25	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
0.625	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
0.313	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
Control	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20

Additional Observations during the Definitive Test

 

All tested loading rates were colourless and visually clear throughout the exposure period. The saturated solution was light orange and visually clear without a Tyndall effect at the start of the exposure (0 hours).

 

 

Water Quality Parameters

 

The measured water quality parameters(i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits. During the test period, the temperature in the incubator was 19 - 20 °C.

 

 

Measured Exposure Concentrations during the Definitive Test

 

The concentrations of the test item Disperse Brown 4 were analytically verified via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the exposure (48 hours) in all tested concentration levels and in the control.

The measured concentrations of the test item were all below the LOQ (0.14 mg/L of the test item) at the start of the exposure (0 hours) and at the end of the exposure period (48 hours).

 

Measured Concentrations and Percent of the Nominal Concentration of the Test ItemDisperse Brown4during the Definitive Test

Sampling date	Fresh media, 0 hours	Old media, 48 hours
Loading rate of the saturated solution [%]	Disperse Brown4
	Meas. conc. [µg/L]	Meas. conc. [µg/L]
10.0	< LOQ	< LOQ
5.00	< LOQ	< LOQ
2.50	< LOQ	< LOQ
1.25	< LOQ	< LOQ
0.625	< LOQ	< LOQ
0.313	< LOQ	< LOQ
Control	< LOQ	< LOQ

Meas. conc.    = measured concentration of the test item, mean value of two injections (control one injection)

LOQ                = limit of quantification (0.140 µg test item/L)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal loading rates of a saturated solution of the test item Disperse Brown 4, the 48-hours EC50 for Daphnia magna was 5.26% (95% confidence limits: 4.55 – 6.75%). The effect level is above the water solubility limit.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Disperse Brown 4 were determined at the test facility according to OECD 202 (2004) from 2019-01-28 to 2019-01-31, with the definitive exposure phase from 2019‑01‑29 to 2019-01-31.

The study was conducted under static conditions over a period of 48 hours with six loading rates (0.313 to 10.0% of the saturated solution) prepared by dilution of a saturated solution with a nominal loading rate of 100 mg/L with dilution water in a geometric series with a separation factor of 2 .All tested loading rates were colourless and visually clear throughout the exposure period. The saturated solution was light orange and visually clear without a Tyndall effect at the start of the exposure (0 hours).

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each dilution level and the control.

The concentrations of the test item Disperse Brown 4 were analytically verified via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the exposure (48 hours) in all tested loading rates and in the control.

The measured concentrations of the test item were all below the LOQ (0.14 mg/L of the test item) at the start of the exposure (0 hours) and at the end of the exposure period (48 hours).

Since the measured concentrations of the test item were below the LOQ, all effect levels given in the table below are based on the nominal loading rates of the test item Disperse Brown 4.

The validity criteria of the test guideline were fulfilled.

 

ELR₁₀-, ELR₅₀- (with 95% Confidence Limits) and ELR₁₀₀-Values

(based on the nominal loading rates of the test item)

Effect levels		Test duration [hours]		DisperseBrown 4 Nominal loading rate of the test item [mg/L]
ELR10 (with 95% confidence limits)		24		4.20		(95% Cl: 3.25 – 5.29)
		48		3.02		(95% Cl: 1.96 – 4.13)
ELR50 (with 95% confidence limits)		24		6.80		(95% Cl: 5.89 – 7.94)
		48		5.26		(95% Cl: 4.55 – 6.75)
ELR100		24		> 10
		48		> 10