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Diss Factsheets

Ecotoxicological information

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 08 21 to 2017-08-24, with the definitive exposure phase from 2017 08 22 to 2017-08-24
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

Constituent 1
Chemical structure
Reference substance name:
2,2'-[[4-[(3,5-dinitro-2-thienyl)azo]phenyl]imino]bisethyl diacetate
EC Number:
255-940-6
EC Name:
2,2'-[[4-[(3,5-dinitro-2-thienyl)azo]phenyl]imino]bisethyl diacetate
Cas Number:
42783-06-2
Molecular formula:
C18H19N5O8S
IUPAC Name:
2,2'-[[4-[(3,5-dinitro-2-thienyl)azo]phenyl]imino]bisethyl diacetate
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.

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 concentration
A saturated solution with a nominal loading of 10.0 mg/L of the test item was tested in a limit test.

Preparation of the Saturated solution
The saturated solution with a nominal loading of 10 mg test item/L was prepared 24 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 hour (1100 rpm) with a magnetic stirrer at room temperature. 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 a limit concentration in the test. During filtration, the filter was always kept covered.

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.

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]: 170
Test temperature:
18 - 22°C, constant within ± 1°C
20.9 °C
pH:
Water Quality Parameters in 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]
10.0* 7.86 7.07
Control 7.72 8.70
* = saturated solution


Water Quality Parameters in 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
10.0* 7.59 8.74 1
Control 7.48 7.81 1
* = saturated solution
Dissolved oxygen:
see above
Conductivity:
Dilution water day 0: 500 [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 concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
other: EL10/50/100
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
other: EL10/50/100
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
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 2017 08-03 to 2017-08-04.

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 1.90 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 1.43 - 2.69 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 analyses for reference item
An EC50-value was calculated for the reference item by sigmoidal dose-response regression. The 95% confidence limits for the EC50 were calculated from the best-fit values, the standard error and the t distribution with the validated 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)

Disperse Blue 284

Nominal loading
of the test item

[mg/L]

IMMOBILIZATION [%]

24 hours

48 hours

Replicates

Replicates

1

2

3

4

MV

1

2

3

4

MV

10.0*

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 ofExposure in the Definitive Test

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

Disperse Blue 284

Nominal loading
of the test item

[mg/L]

Number of immobile Daphnids / Total number of Daphnids

24 hours

48 hours

Replicates

Replicates

1

2

3

4

MV

1

2

3

4

MV

10.0*

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:
In the saturated solution prepared with a nominal loading of 10.0 mg/L of the test item Disperse Blue 284, no effects on Daphnia magna were observed.
Executive summary:

In the acute immobilization test withDaphnia magna (STRAUS), the effects of a saturated solution with a nominal loading of 10.0 mg/L of the test item Disperse Blue 284 were determined at the test facility according to OECD 202 (2004) from 2017‑08‑21 to 2017-08-24, with the definitive exposure phase from 2017‑08‑22 to 2017-08-24.

The limit test was conducted under static conditions over a period of 48 hours.The saturated solution was colourless and 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 item Disperse Blue 284 was analytically verified via HPLC-DAD in fresh media at the start of the exposure (0 hours) in the saturated solution and in the control.

The measured concentration in the saturated solution was < LOD (LOD = 0.03 mg/L with a S/N ratio of 4) 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.

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 itemDisperse Blue 284 in water at the start of the mixing period (loading rate).

The validity criteria of the test guideline were fulfilled.

 

EL10 / 50 / 100-Values (based on the nominal loading of the test item)

Effect values

Test

duration

[hours]

Disperse Blue284

Nominal loading level of the test item

[mg/L]

EL10 / 50 / 100

24

> 10.0

48

> 10.0