Registration Dossier

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 2019-08-28 to 2019-09-13, with the definitive exposure phase from 2019 09-11 to 2019-09-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Determination of the test item
All concentration levels and the control were analytically verified via LC-MS/MS in the fresh media at the start of the exposure (0 hours) as well as in the old media at the end of the exposure (48 hours).
The samples were analysed with an LC-MS/MS method, implemented under non-GLP and documented finally in the GLP raw data.

Sampling for the analytical monitoring
At the start of the exposure (0 hours), samples were taken from the freshly prepared concentration levels and the control for analysis.
At the end of the exposure (48 hours), samples for the analyses of the old media were taken from test replicates containing daphnids.
The samples were immediately stabilized with the solvent methanol (dilution factor 2). They were analyzed on the day of sampling. The samples were stored with the stabilizing solvent at room temperature until analysis, if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
Test item concentrations
Five concentration levels prepared from the stock solution 160 mg/L in a geometric series with a separation factor of 2 by dilution of the stock solution with dilution water, were tested as follows:
10.0 - 20.0 - 40.0 - 80.0 - 160 mg/L.
The test item concentrations were selected based on the results of the first definitive test and a non-GLP preliminary range finding test.

Preparation of the stock solution
The stock solution (160 mg/L of the test item were weighed out) was freshly prepared with dilution water prior to start of the exposure.
The test item solution was mixed thoroughly by manual agitation until it was visually clear.

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, obtained from continuous laboratory cultures.

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guideline.

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. The composition of the culture medium is presented in the table below.

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 are cultured at the test facility


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 to 0.8

Study design

Test type:
static
Water media type:
freshwater
Remarks:
Elendt M4, according to OECD 202, Annex 3 (2004)
Limit test:
no
Total exposure duration:
48 h

Test conditions

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
Salinity:
Not applicable, freshwater
Conductivity:
see any other information on materials and methods incl. tables
Nominal and measured concentrations:
10.0 - 20.0 - 40.0 - 80.0 - 160 mg/L
Details on test conditions:
Test method
The study was performed under static conditions.

Test duration
48 hours

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 each concentration level and the 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. To achieve this, 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 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 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 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

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
147 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: CI: 80.0 - >160
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
85.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: CI: 80.0 - >160
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
156 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: CI: 80.0 - >160
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
113 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: CI: 80.0 - 160
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
160 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 2019 08-07 to 2019-08-08.

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 2.00 mg/L
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000)
Confidence limits 1.00 - 4.00 mg/L


The respective 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 confidence limits.
Reported statistics and error estimates:
Methods of evaluation
The EC100-values after 24 and 48 hours were empirically derived from the observation data. The effect levels (EC10 / 50 / 100) given are based on the analytically verified nominal concentrations of the test item.

EC-values and statistical analyses
The EC10- and the EC50-value after 24 hours were calculated by sigmoidal dose-response regression. The EC10- and the EC50-value after 48 hours were calculated by straight line between the highest concentration resulting in 0% immobilization (EC0) and the test item concentration with the highest immobilization rate. The respective 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 confidence limits. All calculations were carried out from the best-fit values with the software GraphPad Prism. The concentration-effect relationships of the test item are shown graphically.

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

Any other information on results incl. tables

Immobilization Rates after 24 and 48 hours of Exposure in the Definitive Test

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

Nominal test item concentration

[mg/L]       IMMOBILIZATION [%]

      24 hours       48 hours

      Replicates       Replicates

      1       2       3       4       MV       1       2       3       4       MV

160       80       80       80       80       80       100       100       100       100       100

80.0       0       0       0       0       0       0       0       0       0       0

40.0       0       0       0       0       0       0       0       0       0       0

20.0       0       0       0       0       0       0       0       0       0       0

10.0       0       0       0       0       0       0       0       0       0       0

Control       0       0       0       0       0       0       0       0       0       0

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

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

Nominal test item concentration

[mg/L]       NUMBER OF IMMOBILE DAPHNIDS / TOTAL NUMBER OF DAPHNIDS

      24 hours       48 hours

      Replicates       Replicates

      1       2       3       4       Sum       1       2       3       4       Sum

100       4 / 5       4 / 5       4 / 5       4 / 5       16 / 20       5 / 5       5 / 5       5 / 5       5 / 5       20 / 20

50.0       0 / 5       0 / 5       0 / 5       0 / 5       0 / 20       0 / 5       0 / 5       0 / 5       0 / 5       0 / 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

Additional Observations during the Definitive Test

All test item concentrations and the control were colorless and visually clear throughout the exposure period.

Water Quality Parameters

The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits. During the test period, the temperature in the incubator was 21 °C.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Based on the analytically verified nominal concentrations of the test item cyclic Glucamide C8-C10, the 48-hour EC50 for Daphnia magna was 113 mg/L (confidence limits: 80.0 – 160 mg/L).
Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item cyclic Glucamide C8-C10 (batch number: BP 1c (reactor mix)) were determined at the test facility according to OECD 202 (2004) from 2019-08-28 to 2019-09-13, with the definitive exposure phase from 2019 09-11 to 2019-09-13.

The study was conducted under static conditions over a period of 48 hours with five nominal concentrations of the test item cyclic Glucamide C8-C10 prepared in a geometric series with a separation factor of 2 (nominal concentrations of 10 to 160 mg/L).

All test item concentrations and the control were colorless and visually clear throughout the exposure period.

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

The concentrations of the test item cyclic Glucamide C8-C10 were analytically verified via LC-MS/MS 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 all concentration levels and in the control.

The measured concentrations of the test item cyclic Glucamide C8 in the fresh media were in the range of 98 to 104% of the nominal values. At the end of exposure after 48 hours, the measured concentrations were in the range of 97 to 101% of the nominal test item concentrations.

The measured concentrations of the test item cyclic Glucamide C10 in the fresh media were in the range of 96 to 104% of the nominal values. At the end of exposure after 48 hours, the measured concentrations were in the range of 94 to 98% of the nominal test item concentrations.

The measured test item concentrations remained stable within ± 20% of the nominal concentrations over the test period. This indicates that the test item concentrations were successfully maintained for the duration of the test. The effect concentrations are based on the analytically verified nominal concentrations of the test item cyclic Glucamide C8-C10.

The validity criteria of the test guideline were fulfilled.

EC10-, EC50- (with Confidence Limits) and EC100-Values

(based on the nominal concentrations of the test item)

Effect levels      Test duration [hours]       cyclic Glucamide C8-C10 Nominal test item concentrations [mg/L]

EC10 (with confidence limits)       24       147 (Cl: 80.0 – > 160)

                                              48       85.7 (Cl: 80.0 – 160)

EC50 (with confidence limits)       24       156 (Cl: 80.0 – > 160)

                                              48       113 (Cl: 80.0 – 160)

EC100                                     24       > 160

                                               48       160