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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2018-03-20 to 2018-03-22
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:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Determination of the test item
The limit concentration and the control 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).

Sampling for the analytical monitoring
At the start of the exposure (0 hours), samples of the fresh media (control and limit concentration) were taken after preparation of the limit concentration and analyzed. At the end of the exposure (48 hours), samples of the old media (control and limit concentration) were taken directly from the test vessels.

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 limit concentration of 100 mg test item/L was tested. The limit test item concentration was selected based on the results of a non-GLP preliminary range finding test.

Preparation of the stock solution
A stock solution of 100 mg/L was weighed out and freshly prepared with dilutuion water prior to the start of the exposure (at 0 hours). The stock solution was mixed thoroughly by manual agitation.

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. 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:
yes
Total exposure duration:
48 h

Test conditions

Hardness:
Dilution water
0 hours:Total hardness [mg CaCO3/L]: 245
Test temperature:
18 - 22°C, constant within ± 1°C
19 - 20 °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 limit concentration and the control)
Nominal test item concentration
[mg/L] pH-value Dissolved
O2 concentration [mg/L]
100 7.48 9.79
Control 7.45 9.56


Water Quality Parameters in old Media at the End of the Exposure (48 hours)
(measured in one replicate (containing daphnids) of the limit concentration and the control)
Nominal test item concentration
[mg/L] pH-value Dissolved
O2 concentration
[mg/L] Replicate number
100 7.46 9.53 2
Control 7.33 9.35 2


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]
2018-03-20 7.62 9.78 20.2 612 245

Dissolved oxygen:
see above
Conductivity:
Dilution water day 0: 612 [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 concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
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 table below.

EC50-Value (with 95% confidence limits) of the Reference Item Potassium dichromate
based on nominal concentrations mg/L, (0 - 24 hours)
Origin of the daphnids EC50, 24 h
[mg/L] Confidence limits
[mg/L]
Institut für Wasser, Boden und Lufthygiene (WaBoLu) 2.00 1.00 - 4.00





Reported statistics and error estimates:
Methods of evaluation
The effects of the limit concentration were empirically derived from the observation data.
All effect concentrations (EC10 / 50 / 100) given were based on the nominal concentrations 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 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

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In the limit concentration of 100 mg/L of the test item Reactive Orange 12, no effects on Daphnia magna were observed.
Based on the nominal concentrations of the test item Reactive Orange 12, the 48 hours-EC10 / 50 / 100 for Daphnia magna was > 100 mg/L.

No effects were found at the maximum water solubility level in Elendt M4 medium.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effectsof the test item Reactive Orange 12 were determined at the test facility according to OECD 202 (2004) from 2018-03-20 to 2018-03-22.

The limit test was conducted under static conditions over a period of 48 hours.The test solution with a nominal concentration of 100 mg/L showed an orange colour and was visually clearly dissolved throughout the exposure.Twenty daphnids, divided into 4 replicates with 5 daphnids each, were exposed to the limit concentration and the control.

The concentration of the test item Reactive Orange 12 was 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 the limit concentration and in the control.

The measured concentration of the test item in fresh media at the start of the exposure (0 hours)was 100 % of the nominal values. At the end of the exposure period (48 hours), the measured test itemconcentration in the old mediawas 98 % of the nominal values. Since the measured concentrations of the test item Reactive Orange 12 did remain stable within ±20% of the nominal concentrations throughout the exposure period, the nominal concentrations were used for evaluation.

The effect concentrations given in the table below are based on the nominal concentration of the test item Reactive Orange 12.

No effects were found at the limit concentration of 100 mg/L of the test item in Elendt M4 medium.

The validity criteria of the test guideline were fulfilled.

 

EC10 / 50 / 100-Values

(based on the nominal concentration of the test item)

Effect concentrations

Test

duration

[hours]

Reactive Orange 12

Nominal concentration of the test item

[mg/L]

EC10 / 50 / 100

24

> 100

48

> 100