Methyl N-[3-(acetylamino)-4-[(2,4-dinitrophenyl)azo]phenyl]-N-(3-methoxy-3-oxopropyl)-β-alaninate

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 2018-04-03 to 2018-04-06, with the definitive exposure phase from 2018-04-04 to 2018-04-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

Methyl N-[3-(acetylamino)-4-[(2,4-dinitrophenyl)azo]phenyl]-N-(3-methoxy-3-oxopropyl)-β-alaninate

EC Number:

274-828-8

EC Name:

Methyl N-[3-(acetylamino)-4-[(2,4-dinitrophenyl)azo]phenyl]-N-(3-methoxy-3-oxopropyl)-β-alaninate

Cas Number:

70729-65-6

Molecular formula:

C22H24N6O9

IUPAC Name:

methyl N-[3-(acetylamino)-4-[(2,4-dinitrophenyl)azo]phenyl]-N-(3-methoxy-3-oxopropyl)-β-alaninate

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analytically verified via HPLC-DAD in fresh media at the start of the exposure (0 hours). The measured concentration in the highest concentrated sample was close to LOD (LOD = 0.05 mg/L with a S/N ratio of 3) but lower the lowest calibration standard. All other samples were < LOD 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 considered to be below the LOD in the 48-hours old media, too.

Sampling for the analytical monitoring
At the start of the exposure (0 hours), samples of the fresh media (control and all concentration levels) were taken after preparation of the test item concentrations and analyzed.

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

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the saturated solution
A saturated solution (100 mg/L test item was weighed out) was prepared with dilution water 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 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 as the highest concentration level and as a stock solution for the preparation of further dilution levels by dilution with dilution water. 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 observed to be negative.

Test item loadings
6 loading levels 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:
0.0977 - 0.391 - 1.56 - 6.25 - 25.0 - 100% of the saturated solution.
The test concentrations are 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).

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. 1500 lx

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004) is used.

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 +/- 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, 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

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]: 237

Test temperature:

18 - 22°C, constant within ± 1°C
20 °C

pH:

Water Quality Parameters in fresh Media at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) per concentration level and control)
Dilution Level of the Saturated solution
[%] pH-value Dissolved
O2 concentration [mg/L]
100 7.69 8.58
25.0 7.67 9.33
6.25 7.67 9.88
1.56 7.65 9.96
0.391 7.65 9.97
0.0977 7.63 9.96
Control 7.70 9.87


Water Quality Parameters in old Media at the End of the Exposure (48 hours)
(measured in the replicate with the highest immobilization rate (containing daphnids) per concentration and control)
Dilution level of the saturated solution
[%] 48 hours
pH-value Dissolved
O2 concentration
[mg/L] Replicate number
100 7.90 7.65 3
25.0 7.81 7.62 3
6.25 7.99 7.83 3
1.56 7.72 7.91 3
0.391 7.85 8.00 3
0.0977 7.98 7.97 3
Control 8.01 7.98 3


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]
2018-04-04 7.86 10.06 19.6 618 237

Dissolved oxygen:

see above

Conductivity:

Dilution water day 0: 618 [µS/cm]

Details on test conditions:

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

Test volume
20 mL

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 the limit concentration and 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. 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 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 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 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

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EL100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EL100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

Test of the Reference Item

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 2018-03-13 to 2018-03-14. For results of the most recent of the monthly performed reference tests, see the table below.

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 2.00 mg/L (1 – 4 mg/L)
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000)
0.6 - 2.1 mg/L, acc. to OECD 202 (2004)

Reported statistics and error estimates:

Methods of evaluation
The EL100 (after 24 and 48 hours) was derived empirically from the observation data. For determination of the EL10 / 50 (after 48 hours) a sigmoidal dose-response test was done. An EL50 could not be derived, while an EL10 of 95 mg/L was found by sigmoidal-dose-response (ambigious result, documented in the raw data, data not shown). Since the sigmoidal-dose response analysis was found to be ambigious, a Normality test (Shapiro-Wilk) was performed and failed, followed by a Kruskal-Wallis One Way Analysis of Variance. Since results after 24 hours and 48 hours show no difference, only results after 48 hours were evaluated.

EC-values and statistical analyses
The EC50-value for the reference item and its 95% confidence limits 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.
- SigmaPlot, SPSS INC.
- Excel, MICROSOFT CORPORATION

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal loading concentrations of the test item Disperse Red 311, the 48 hour-EC50 for Daphnia magna was >100 % of the saturated solution, corresponding to a nominal concentration of 100 mg test item/L.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Disperse Red 311 were determined at the test facility according to OECD 202 (2004) from 2018-04-03 to 2018-04-06, with the definitive exposure phase from 2018-04-04 to 2018-04-06.

The study was conducted under static conditions over a period of 48 hours with six concentration levels prepared by dilution from a saturated solution of the test item (nominal loading 100 mg/L). The tested concentration levels were0.0977 - 0.391 - 1.56 - 6.25 - 25.0 - 100 % of the saturated solutionwith a separation factor of 4.

The test itemis a red solid with awater solubility of <0.024 mg/L (OECD 105). The test item concentrations were visually clear and concentration related violet colored throughout the exposure period.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration leveland the control.

The concentrationsof the test item were analytically verified via HPLC-DAD in fresh media at the start of the exposure (0 hours) in all concentration levels and in the control.

The measured concentration in the highest concentrated sample was close to LOD (LOD = 0.05 mg/L with a S/N ratio of 3) but lower the lowest calibration standard. All other samples were < LOD 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 considered to be below the LOD in the 48-hours old media, too.

All effect concentration levels (EL_{10 / 50 / 100}) given in the table below are based on the dilution levels of the saturated solution of the test itemDisperseRed 311.

The validity criteria of the test guideline were fulfilled.

 

EL₁₀-, EL₅₀-and EL₁₀₀-Values

(based on the dilution level of the saturated solution of the test item)

Disperse Red 311
Effect concentrations	Test duration [hours]	Dilution Level of the Saturated Solution [%]
EL10	24	>100*
	48	>100*
EL50	24	>100
	48	>100
EL100	24	>100
	48	>100

* = Evaluation by sigmoidal-dose response analysis gave an EL₁₀of 95 mg/L. No statistical difference between the test

      concentrations was found by Kruskal-Wallis ANOVA, therefore the result by sigmoidal-dose response is considered not reliable.