2-[[4-[(2-cyanoethyl)(2-phenylethyl)amino]phenyl]azo]-5-nitrobenzonitrile

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:

from 2017-11-27 to 2017-11-30, with the definitive exposure phase from 2017-11-28 to 2017-11-30

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-[2-[(2,6-dicyano-4-nitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

EC Number:

255-473-8

EC Name:

N-[2-[(2,6-dicyano-4-nitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

Cas Number:

41642-51-7

Molecular formula:

C20H19N7O3

IUPAC Name:

N-{2-[(2,6-dicyano-4-nitrophenyl)diazenyl]-5-(diethylamino)phenyl}acetamide

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The saturated solution and the control were analytically verified via
HPLC-DAD in fresh media at the start of the exposure period (0 hours). Since the measured test item concentration was even below the LOD in fresh media at the start of the exposure period, the analytical monitoring at the end of the exposure was waived and the test item concentration was considered to be below the LOD in the 48 hours old media, too. Details of the analytical method are presented in section 12. Analytical results are presented in section 7.1.3.

Sampling for the analytical monitoring
At the start of the exposure (0 hours), samples of the fresh media
(control and saturated solution) were taken after preparation of the saturated solution and analyzed.

Criteria for the analytical monitoring (target)
Recoveries of the test item should be within ± 20% of the nominal or
initially measured concentration.

Test solutions

Vehicle:

no

Details on test solutions:

Limit loading level
A saturated solution with a nominal loading of 100 mg/L of the test item was tested in a limit test.
The limit loading level was selected based on the results of a non-GLP preliminary range finding test. For results, see Annex I.

Preparation of the saturated solution
A saturated solution (100 mg/L test item were weighed out) was
prepared with dilution water (see Table 2) one day before the start of the exposure (at -24 hours).
The test item was mixed with the dilution water by constant stirring with a magnetic stirrer at approximately 1100 rpm for 24 hours at room temperature. After completion of stirring, 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 were discarded. The following filtrate, i.e. the saturated solution, was tested in a limit test. During filtration, the filter was always kept covered.
The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.

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

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
Daphnia magna STRAUS (Clone 5).

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

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. 20 µEm-2  s-1

Culture medium
Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L, was used. The composition of the culture medium is presented in Table 2.

Culture feeding
The culture 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, with an algae cell density of > 106 cells/mL. The algae were cultured at the test facility.

Origin of the food algae
Sammlung von Algenkulturen (SAG),
Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany


Composition of the Culture Medium according to ELENDT (1990)
Component Concentration [mg/L]
CaCl2 x 2 H2O 147
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 4.30
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18
Na2EDTA x 2 H2O 5.00
FeSO4 x 7 H2O 1.99
H3BO3 0.29
MnCl2 x 4 H2O 0.36
LiCl 0.30
SrCl2 x 6 H2O 0.15
RbCl 0.071
NaBr 0.016
Na2MoO4 x 2 H2O 0.063
CuCl x 2 H2O 0.017
ZnCl2 0.013
CoCl2 x 6 H2O 0.010
KJ 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamine 0.0010
Biotin 0.00075
pH 8.2  0.8

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

Dilution water
0 hours:Total hardness [mg CaCO3/L]: 180

Test temperature:

18 - 22°C, constant within ± 1°C
20.5 °C

pH:

Water Quality Parameters in the fresh Media at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) of the saturated solution and the control)
Nominal loading
of the test item
[mg/L] pH-value Dissolved
O2 concentration [mg/L]
100* 7.83 6.51
Control 7.79 9.00
* = saturated solution


Table : Water Quality Parameters in the old Media at the End of the Exposure (48 hours)
(measured in one replicate (containing daphnids) of the saturated solution and the control)
Nominal loading
of the test item
[mg/L] pH-value Dissolved
O2 concentration
[mg/L] Replicate number
100* 7.68 8.51 1
Control 7.58 8.54 1
* = saturated solution


Table : Water Quality Parameters of the Dilution Water at the Start of the Exposure (0 hours)
Dilution water
dated: pH-value

Dissolved
O2 concentration [mg/L] Temperature

[°C] Conductivity

[µS/cm] Total hardness

[mg CaCO3/L]
2017-11-28 7.79 9.00 20.5 478 180

Dissolved oxygen:

see above

Conductivity:

Dilution water day 0: 478 [µS/cm]

Details on test conditions:

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

Age of the daphnidsat the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for
the study. 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 of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

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

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

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

other: EL10/50/100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

other: EL10/50/100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

A reference test was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in Elendt M4 medium (Table 2) under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility. The results of the most recent test are presented in section 7.2.

Reference item Potassium dichromate p.a. (SIGMA-ALDRICH)

Purity 99.0%

Batch number MKBV0900V

Expiry date 2021-11-25

Test concentrations 1.00 – 2.00 – 4.00 mg/L

Ranges of validity EC50 (24 hours): 0.6 - 2.4 mg/L, according to AQS P 9/2 (clone 5)
EC50 (24 hours): 0.6 - 2.1 mg/L, according to OECD 202 (clone A)

Exposure phase 2017-11-02 to 2017-11-03

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 2017 11-02 to 2017-11-03. For results of the most recent of the monthly performed reference tests, see Table 6.

Table 6: EC50-Value (with 95% Confidence Limits) of the Reference Item Potassium dichromate
based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 2.10 mg/L
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A
95% confidence limits 1.92 - 2.43 mg/L

Reported statistics and error estimates:

Methods of evaluation No calculations were made, because no effects on Daphnia magna were observed.
According to OECD Guidance Document No 23 (2000), if an effect cannot be detected in a test with a saturated solution, the result has to be reported as no toxic effects at saturation. Therefore, all effect levels (EL10 / 50 / 100) given were based on the nominal loading of the test item.

EC-values and statistical An EC50-value was calculated for the reference item by sigmoidal
analyses for reference item dose-response regression. 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 Prism5.

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 Prism5, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

The concentration of the test item DisperseBlue 165 was analytically verified via HPLC-DAD in the fresh media at the start of the exposure (0 hours) in the saturated solution and in the control. Details of the analytical method are presented in section 12.

The measured concentration in the saturated solution was < LOD (0.00625 mg/L with a S/N ratio of 3) at the start of the exposure (0 hours). Therefore, analytical monitoring at the end of the exposure was waived and the test item concentration was considerd to be below the LOD in the 48-hours media, too.For results, seeTable5.

Since the recoveries at 0 hours were < LOD, the method validation was not necessary.

 

Table5:      Measured Concentrations ofDisperseBlue 165in the Fresh Media at 0 hours

Sampling date:	0 hours (fresh media)
Nominal loading of the test item in the saturated solution [mg/L]	DisperseBlue165
	Meas. conc. [mg/L]
100	< LOD
Control	< LOD

Meas. conc.    = measured concentration of the test item, mean value of 2 injections

LOD                 = limit of detection (0.00625 mg/L)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In the saturated solution prepared with a nominal loading of 100 mg/L of the test item Disperse Blue 165, no effects on Daphnia magna were observed.

Executive summary:

In the acute immobilization test with Daphnia magna(STRAUS), the effectsof a saturated solution with a nominal loading of100 mg/Lof the test itemDisperseBlue 165 were determined at the test facility according to OECD 202 (2004) from2017‑11‑27 to 2017-11-30, with the definitive exposure phase from 2017‑11‑28 to 2017-11-30.

The limit test was conducted under static conditions over a period of 48 hours.The saturated solution (prepared as specified in section4.2) was visually clear during the exposure period.Twenty daphnids, divided into 4 replicates with 5 daphnids each, were exposed to the saturated solution and the control.

The concentration of the test itemDisperseBlue 165was analytically verified via HPLC-DAD in the fresh media at the start of the exposure (0 hours) in the saturated solution and in the control. Details of the analytical method are presented in section 12.

The measured concentration in the saturated solution was < LOD (0.00625 mg/L with a S/N ratio of 3) at the start of the exposure (0 hours). Therefore, analytical motoring at the end of the exposure was waived and the test item concentration was considerd to be below the LOD in the 48-hours media, too.For results, seeTable5.

According to OECD Guidance Document No 23 (2000), if an effect cannot be detected in a test with a saturated solution, the result has to be reported as no toxic effects at saturation.Exposures are expressed in terms of the original concentration of the test itemDisperseBlue 165in water at the start of the mixing period (loading rate). The effect values are presented inTable1.

The validity criteria of the test guideline were fulfilled.

 

Table1:   EL_{10 / 50 / 100}-Values

(based on the nominal loading of the test item)

Effect values	Test duration [hours]	Disperse Blue165 Nominal loading level of the test item [mg/L]
EL10 / 50 / 100	24	> 100
	48	> 100