Methyl N-ethyl-N-[4-[(5-nitro-2,1-benzisothiazol-3-yl)azo]phenyl]-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 2017-07-24 to 2017-07-27, with the definitive exposure phase from 2017-07-25 to 2017 07-27

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

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:

Determination of the test item
All tested loading levels 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 concentrations were 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.

Sampling for the analytical monitoring
At the start of the exposure (0 hours), samples of the fresh media were taken after preparation of all loading levels 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:

Preparation of the saturated solution
The 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 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. No Tyndall effect was observed. Thus, no undissolved test item was present.

Test loadings
5 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 (see Table 1), were tested as follows: 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
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. 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:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Dilution water dated:
0 hours:Total hardness [mg CaCO3/L]: 178

Test temperature:

18 - 22°C, constant within ± 1°C
20.8 °C

pH:

Water Quality Parameters in fresh Media at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) per loading level and control)
Nominal loading level
of the test item
[%] pH-value Dissolved
O2 concentration [mg/L]
100* 8.40 7.44
50.0 8.47 8.08
25.0 8.48 8.52
12.5 8.44 8.64
6.25 8.36 8.57
Control 8.47 8.67
* = saturated solution

Water Quality Parameters in old Media at the End of the Exposure (48 hours)
(measured in one appropriate replicate (containing daphnids) per loading level and control)
Nominal loading level
of the test item
[%] pH-values Dissolved
O2 concentration
[mg/L] Replicate number
100* 7.64 7.50 3
50.0 7.62 7.25 1
25.0 7.69 7.56 1
12.5 7.72 7.75 1
6.25 7.82 8.02 1
Control 7.81 7.89 1
* = saturated solution

Dissolved oxygen:

see above

Conductivity:

Dilution water day 0: 441 [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

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

67.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI:59.8 - 77.5

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 of exposure from 2017 07-11 to 2017-07-12.

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 1.31 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 1.05 - 1.72 mg/L

Reported statistics and error estimates:

Methods of evaluation
The EL50-value after 48 hours and the EL100-values (after 24 and 48 hours) were empirically derived from the observed immobilization rates. All effect levels (EL10 / 50 / 100) given are based on the nominal loadings of the test item, since the test item could not be detected. The loading-effect relationships of the test item after 24 and 48 hours are shown graphically.


EL-/EC-values and statistical analyses
The EL10- and the EL50-values with 95% confidence limits after 48 hours were calculated by sigmoidal dose-response regression. The loading-effect relationship is shown graphically. The EC50-value with 95% confidence limits for the reference item was calculated accordingly by sigmoidal 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 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 Prism5, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

Immobilization Ratesafter 24 and 48 hours ofExposure in the Definitive Test

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

DisperseBlue 148 Nominal loading level of the test item [%]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100*	0	20	20	0	10	60	80	100	80	80
50.0	20	0	0	0	5	40	0	20	40	25
25.0	0	0	0	0	0	0	0	0	0	0
12.5	0	0	0	0	0	0	0	0	0	0
6.25	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

*    = saturated solution

 

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

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

DisperseBlue 148 Nominal loading level of the test item [%]	Number of immobile Daphnids / Total number of Daphnids
	24 h					48 h
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100*	0 / 5	1 / 5	1 / 5	0 / 5	2 / 20	3 / 5	4 / 5	5 / 5	4 / 5	16 / 20
50.0	1 / 5	0 / 5	0 / 5	0 / 5	1 / 20	2 / 5	0 / 5	1 / 5	2 / 5	5 / 20
25.0	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
12.5	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
6.25	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

*    = saturated solution

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal loading levels of the test item Disperse Blue 148, the 48 hour-EL50 for Daphnia magna was 67.9 % of the saturated solution (95% confidence limits: 59.8 – 77.5 %). All effects are far above the water solubility limit of the test item.

Executive summary:

In the acute immobilization test to Daphnia magna (STRAUS), the effects of the test item Disperse Blue 148 were determined at the test facility according to OECD 202 (2004) from2017-07-24 to 2017-07-27, with the definitive exposure phase from 2017-07-25 to 2017‑07-27.

The study was conducted under static conditions over a period of 48 hours with a saturated solution with a nominal loading of 100 mg/Lof the test itemDisperse Blue 148and further four dilution levels in a geometric series with a separation factor of2(6.25 to 50% of the saturated solution).All tested concentration levels were visually clear throughout the exposure period. The two highest loading levels (50 and 100% of the saturated solution) were slightly grey - blue at the start of the exposure (0 hours).

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

The concentrations of the test item Disperse Blue 148 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 concentrations of the main component of the test item were below (LOD = 0.04 mg/L with a S/N ratio of 5) at the start of the exposure (0 hours) in all loading levels. Therefore, analytical monitoring at the end of the exposure was waived and the test item concentrations were considered to be below the LOD in the 48-hours old media, too.

All effect levels given are based on the nominal loading levels of the test itemDisperseBlue 148.

The validity criteria of the test guideline were fulfilled.

 

 EL₁₀-, EL₅₀- (with 95% Confidence Limits) and EL₁₀₀-Values

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

DisperseBlue 148
Effect levels		Test duration [hours]		Nominal loading level of the test item [%]
EL10 (with 95% confidence limits)		24		98.4		(Cl: 52.8 – > 100)
		48		37.7			(Cl: 29.0 – 47.0)
EL50 (with 95% confidence limits)		24		> 100		(Cl: Not applicable)
		48		67.9		(Cl: 59.8 – 77.5)
EL100		24		> 100
		48		> 100