Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2018-05-27 to 2018-06-01, with the definitive exposure phase from 2018-05-30 to 2018-06-01
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Determination of the test item
The limit concentration and the control were analytically verified via LC-MS/MS in fresh media at the start of exposure and at water renewal (0 and 24 hours) and in 24-hours old media at renewal and at the end of the exposure (24 and 48 hours). The samples were analysed with an LC-MS/MS method, implemented under non-GLP and documented finally in the GLP raw data. The method was validated.

Sampling for the analytical monitoring
At the start of the exposure and at renewal (0 and 24 hours), samples of the fresh media were taken after preparation of the saturated solution and analyzedAt renewal and at the end of the exposure (24 and 48 hours), samples of the 24-hours old media were taken directly from the test vessels and analyzed.

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

Vehicle:
no
Details on test solutions:
Limit concentration
A saturated solution prepared with a nominal concentration of 100 mg/L of the test item was tested. The limit concentration is based on the results of a non-GLP preliminary range finding test.

Preparation of the saturated solution
A saturated solution of 100 mg test item/Lwas prepared in dilution water three days before the start of the exposure (at day -3) and three days before the renewal of the test solutions (at day -2).
A glass bottle was filled with an appropriate amount of the dilution water, an appropriate volume of the test item (density: 0.98 g/cm³ (20 °C) was taken into account) was placed by pipette onto the water surface and the bottle was closed with the screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out for 72 hours with a magnetic stirrer 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 test item solution, was used as 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 groups.

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. The algae were cultured at the test facility.

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
see any other information on materials and methods incl. tables
Test temperature:
see any other information on materials and methods incl. tables
pH:
see any other information on materials and methods incl. tables
Dissolved oxygen:
see any other information on materials and methods incl. tables
Conductivity:
see any other information on materials and methods incl. tables
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
Duration:
24 h
Dose descriptor:
other: EC10/50/100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
other: EC10/50/100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Results with reference substance (positive control):
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 05-03 to 2018-05-04. For results of the most recent of the monthly performed reference tests, see the table below.

EC50-Value (with confidence limits) of the Reference Item Potassium dichromate
based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 2.07 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


Reported statistics and error estimates:
Methods of evaluation
The effects of the limit concentration were deduced directly from the observed immobilization rates.
Since the measured concentrations were < LCL, the effect levels (EC10 / 50 / 100) were based on the nominal concentration of the test item.

ECx-values and statistical analyses for reference item
An EC50-value was calculated for the reference item by linear dose-response regression. The respective 95% confidence limits were empirically derived from the observation data as follows: The highest concentration level without any effect (EC0) and the lowest concentration level causing 100% immobilization (EC100) were used as 95% confidence limits. 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



Preliminary Range Finding Test (non-GLP)

A non-GLP preliminary range finding test was conducted at the test facility under semi-static conditions over a period of 48 hours with threedilution levels 1, 10 and 100 % of a saturated solution prepared as specified in section 4.2 for the definitive test with dilution water. The dilution levels 1 and 10% were prepared by dilution of the saturated solution with dilution water. The saturated solution and all dilution levelswere colorless and visually clear throughout the exposure period.

The preliminary range finding test was conducted under diffuse light conditions (light intensity of max. 1500 lx, 16/8 hours light/dark cycle).

In the range finding test, two replicates per dilution level and control, each with five daphnids, were tested.

 

Immobilization Rates in the non-GLP Preliminary Range Finding Test

                (n = 10, divided into 2 replicates with 5 daphnids each)

Dilution level

of the saturated

solution

[%]

IMMOBILIZATION [%]

24 hours

48 hours

Replicates

Replicates

1

2

MV

1

2

MV

100*

0

0

0

0

40**

20

 10

0

0

0

0

0

 0

   1

0

0

0

0

0

 0

Control

0

0

0

0

0

 0

*      = saturated solution

**     = considered as an outlier, since no effects were observed in the 2ndreplicate

 

Measured Concentrations of Reaction products of alcohols,C11-14-iso,C13 rich and phosphorus pentoxide during the non-GLP Preliminary Range Finding Test

                Analytical system: LC-MS/MS

Sampling date

Fresh media,

0 hours

Old media,

24 hours

Fresh media,

24 hours

Old media,

48 hours

Dilution level

of the saturated

solution

[%]

Reaction products of alcohols,C11-14-iso,C13 rich and phosphorus pentoxide

Meas. conc.

[µg/L]

Meas. conc.

[µg/L]

Meas. conc.

[µg/L]

Meas. conc.

[µg/L]

100

< LCL

< LCL

< LCL

< LCL

 10

< LCL

< LCL

< LCL

< LCL

   1

< LCL

< LCL

< LCL

< LCL

Control

< LCL

< LCL

< LCL

< LCL

Meas. conc.    = measured concentration of the test item, singledeterminations, dilution factors taken into account

LCL        = lowest calibration Level (80 µg/L of the test item)

Validity criteria fulfilled:
yes
Conclusions:
In the limit concentration 100 mg/L of the test item Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide, no effects on Daphnia magna were observed.
Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide were determined at the test facility according to OECD 202 (2004) from 2018-05-27 to 2018-06-01, with the definitive exposure phase from 2018-05-30 to 2018-06-01.

The study was conducted under semi-static conditions over a period of 48 hours with the limit concentration of 100 mg/L of the test item Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide.The limit concentration was prepared by slow stirring for 72 hours and subsequent filtration.The limit concentrationwas visually clear and colourless 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 itemReactionproducts of alcohols, C11-14-iso,C13 rich and phosphorus pentoxidewas analytically verified via LC-MS/MS in fresh media of the limit concentration and in the control at the start of the exposure and at renewal (0 and 24 hours) and in 24-hours old media at renewal and at the end of the test (24 and 48 hours).

The measured concentration of the test item in fresh media at the start and the end of the exposure intervals was < LCL.

Since the measured test item concentration was < LCL, the effect concentrations given based on nominal concentrations of the test item Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide.

The validity criteria of the test guidelines were fulfilled.

 

 

In the limit concentration100 mg/Lof the test item Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide, no effects on Daphnia magna were observed.

 

 

 

EC10-, EC50-, EC100-Values

(based on the nominal concentration of the test item)

Effect concentrations

 

 

Test

duration

[hours]

Toxicity endpoint
based on the nominal concentration the test item

[mg/L]

EC10 / 50 / 100

24

> 100

48

> 100

Description of key information

In the limit concentration 100 mg/L of the test item Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide, no effects on Daphnia magna were observed.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information