Registration Dossier

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 2016-07-12 to 2016-07-15, with the definitive exposure phase from 2016-07-13 to 2016-07-15
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:
C10-13-alkyl(branched, unsaturated)-succinic acid-4-(3-methyl-butyl)-ester
Molecular formula:
C18H32O4 - C22H40O4
IUPAC Name:
C10-13-alkyl(branched, unsaturated)-succinic acid-4-(3-methyl-butyl)-ester
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 UPLC-DAD at the start (0 hours) and at the end of the exposure (48 hours). The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Sampling for the analytical monitoring
At the start of the exposure (0 hours), sampling was carried out after preparation of the test item concentrations. At the end of the exposure (48 hours), samples were taken directly from the test vessels.

Quality criteria
Recoveries of the test item should be within ± 20% of the nominal or initially measured concentrations.

Test solutions

Vehicle:
no
Details on test solutions:
Preparation of the saturated solution
A saturated solution with a nominal concentration of 193 mg/L of the
test item, which corresponds to 199 µL/L (density taken into account) of the test item, was prepared one day prior to the start of the exposure (at -24 hours) in a glass flask with a lateral outlet (nominal capacity: 1 L).
The glass flask was filled with 1 L of the dilution water. The test item (199 µL) was placed by pipette onto the water surface. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out for 24 ± 1 hours with a magnetic stirrer at room temperature. After completion of stirring, the saturated solution was allowed to stand for 1 hour for separation of undissolved test item. The saturated solution was removed by the lateral outlet of the flask (from the approximate bottom of the glass flask). Due to the density of the test item, any undissolved material was expected to float on the surface. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of emulsion), which could not be observed. 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.

Test concentrations / solutions
5 test item concentrations in a geometric series with a separation factor of 2, prepared by diluting the saturated solution with dilution water, were tested as follows: 6.25 - 12.5 - 25 - 50 - 100% of the saturated solution
The test concentrations 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 selectionof 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), modified to a total hardness of 160 to 180 mg CaCO3/L, is 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

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
see any other information on material and methods incl. tables


Test temperature:
see any other information on material and methods incl. tables
pH:
see any other information on material and methods incl. tables
Dissolved oxygen:
see any other information on material and methods incl. tables





Conductivity:
see any other information on material and methods incl. tables
Nominal and measured concentrations:
Nominal: 6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution
Geometric mean measured test item concentrations: 1-30 - 2.18 - 3.68 - 6.66 - 13.9 mg/L.
Details on test conditions:
Test method
Based on the results of a preliminary range finding test, the study was performed with a static test design, since the measured concentrations remained stable over 48 hours under test 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 in each test replicate (see ‘Application’)

Dilution water
Same as culture medium

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 daphnids
Less than 24 hours old daphnids from a healthy stock were used for at the start of the exposure 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 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

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
6.64 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: CI: 5.16 - 8.66
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
6.22 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: CI: < 2.18 - 11.2
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
9.76 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: CI: 8.39 - 11.2
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
8.03 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: CI: 6.90 - 12.0
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 13.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
13.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
The concentrations of the test item Tetrapropylene succinic acid monoisobutylester were analytically verified via HPLC-DAD at the start (0 hours) and at the end of the exposure (48 hours) in all concentration levels and in the control.
The measured concentrations of the test item were in the range of 85 to 102% of the 0 hour measured concentrations at 48 hours at the biologically most relevant concentration levels of 12.5 to 100% of the saturated solution. The analytical results reflected the dilution factor of 2 between the concentration levels. At the two lowest concentration levels 6.25 and 12.5% of the saturated solution, the measured values were below the LOQ. Since the concentration level of 6.25% of the saturated solution was below the EC0, it was excluded from further evaluation. At the concentration level of 12.5% of the saturated solution, the measured concentrations were > 70% of the LOQ and were therefore used for the evaluation.
The measured test item concentrations remained stable within ± 20% of the 0 hour measured concentrations at 48 hours at the biologically most relevant concentration levels of 12.5 to 100% of the saturated solution. This indicates that the test item concentrations were successfully maintained for the duration of the test. The effect levels given are based on the geometric mean measured concentrations of the test item Tetrapropylene succinic acid monoisobutylester.
The geometric mean measured concentrations of the biologically most relevant concentration levels were determined to be 2.18 - 3.68 - 6.66 - 13.9 mg/L.
Results with reference substance (positive control):
The percentage of immobility for the reference item was determined after 24 hours. The EC50-value with 95% confidence limits (CI) was calculated by sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was:
EC50: 2.00 mg/L (Cl: 1.22 - 3.29 mg/L)
The EC50-value of the reference item potassium dichromate after 24 hours is within the required concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.
Reported statistics and error estimates:
Methods of evaluation
The EC100-values after 24 and 48 hours were estimated directly from the observation data.
The effect levels (EC10 / 50 / 100) given are based on the geometric mean measured concentrations of the test item.

EC-values and statistical analyses
The EC10- and the EC50-values after 24 and 48 hours were calculated 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 Prism5. The concentration-effect relationships of the test item are shown graphically.
The EC50-value for the reference item and its confidence limits were calculated accordingly.

Software alculation
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)

Geometric mean measured concentration of the test item

[mg/L]

IMMOBILIZATION [%]

24 hours

48 hours

Replicates

Replicates

1

2

3

4

MV

1

2

3

4

MV

13.9

80

80

100

100

90

100

100

100

100

100

 6.66

 0

 0

 20

 20

10

 20

   0

 20

 20

 15

 3.68

20

 0

 0

 0

 5

 40

   0

   0

   0

 20

 2.18

 0

 0

   0

   0

 0

   0

   0

   0

   0

   0

 1.30*

 

 

   

   

 

   

   

   

   

   

Control

 0

 0

   0

   0

 0

   0

   0

   0

   0

   0

* = concentration excluded from evaluation (below EC0and measured concentrations < 70% of the LOQ)

 

 Measured Exposure Concentrations during the Definitive Test

 

The concentrations of the test itemTetrapropylenesuccinic acid monoisobutylester were analytically verified via HPLC-DAD at the start (0 hours) and at the end of the exposure (48 hours) in all concentration levels and in the control. The signal was confirmed by a NON-GLP LC-MS/MS analysis (details given in a Statement of Noack Laboratories: 151110CB / CKK16906).

The measured concentrations of the test item were in the range of 85 to 102% of the 0 hour measured concentrations at 48 hours at the biologically most relevant concentration levels of 12.5 to 100% of the saturated solution. The analytical results reflected the dilution factor of 2 between the concentration levels. At the two lowest concentration levels 6.25 and 12.5% of the saturated solution, the measured values were below the LOQ. Since the concentration level of 6.25% of the saturated solution was below the EC0,it was excluded from further evaluation. At the concentration level of 12.5% of the saturated solution, the measured concentrations were > 70% of the LOQ and were therefore used for the evaluation.

The measured test item concentrations remained stable within±20% of the 0 hour measured concentrations at 48 hours at the biologically most relevant concentration levels of 12.5 to 100% of the saturated solution. This indicates that the test item concentrations were successfully maintained for the duration of the test.

The geometric mean measured concentrations of the biologically most relevant concentration levels were determined to be 2.18 - 3.68 - 6.66 - 13.9 mg/L.

 

Table6:      Measured Concentrations of the Test ItemTetrapropylenesuccinic acid monoisobutylesterduring the Definitive Test

Sampling date

2016-07-13

Start of the exposure,
0 hours

2016-07-15

End of the exposure,
48 hours

 

Start of analysis

2016-07-13

2016-07-15

 

Nominal concentration of

the saturated solution

[%]

Tetrapropylenesuccinic acid monoisobutylester

Geometric mean

measured concentration

[mg/L]

Meas. conc.

[mg/L]

Meas. conc.

[mg/L]

%

100

13.8

14.1

102

13.9

 50

 6.70

 6.63

 99

  6.66

 25

 3.98

 3.40

 85

 3.68

 12.5

 2.181)

 2.191)

100

 2.181)

   6.25

< LOQ

< LOQ

 1.302)

Control

< LOQ

< LOQ

 

Meas. conc.= measured concentration of the test item, mean value of 2 injections, dilution factors taken into account

%                  = percent of the initially measured concentration of the test item

LOQ             = limit of quantification of the analytical method (2.60 mg/L of the test item)

1)                   = < LOQ, but > 70% LOQ

2)                   = set to ½ of the LOQ, since measured values were < 70% of the LOQ

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Based on the geometric mean measured concentrations of the test item, the 48 hour-EC50 for Daphnia magna was 8.03 mg/L (95% confidence limits: 6.90 – 12.0 mg/L).
Executive summary:

In the acute immobilization test withDaphnia magna (STRAUS), the effects of the test item were determined at the test facility according to OECD 202 (2004) from2016-07-12 to 2016-07-15, with the definitive exposure phase from 2016-07-13 to 2016-07-15.

The study was conducted under static conditions over a period of 48 hours with a saturated solution (prepared with a nominal concentration of 199 µL/Lof the test itemTetrapropylenesuccinic acid monoisobutylester) and further four dilution levels prepared in a geometric series with a separation factor of2(nominal concentrations of6.25 to 100%of the saturated solution).

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

The concentrations of the test itemTetrapropylenesuccinic acid monoisobutylesterwere analytically verified via HPLC-DAD at the start (0 hours) and at the end of the exposure (48 hours) in all concentration levels and in the control. Details of the analytical method are presented in section14.

The measured concentrations of the test item were in the range of 85 to 102% of the 0 hour measured concentrations at 48 hours at the biologically most relevant concentration levels of 12.5 to 100% of the saturated solution. The analytical results reflected the dilution factor of 2 between the concentration levels. At the two lowest concentration levels 6.25 and 12.5% of the saturated solution, the measured values were below the LOQ. Since the concentration level of 6.25% of the saturated solution was below the EC0,it was excluded from further evaluation. At the concentration level of 12.5% of the saturated solution, the measured concentrations were > 70% of the LOQ and were therefore used for the evaluation.The analytical results arepresented inTable 6.

The measured test item concentrations remained stable within±20% of the 0 hour measured concentrations at 48 hours at the biologically most relevant concentration levels of 12.5 to 100% of the saturated solution. This indicates that the test item concentrations were successfully maintained for the duration of the test. The effect levels given are based on the geometric mean measured concentrations of the test itemTetrapropylenesuccinic acid monoisobutylester.

The geometric mean measured concentrations of the biologically most relevant concentration levels were determined to be 2.18 - 3.68 - 6.66 - 13.9 mg/L.

The validity criteria of the test guideline were fulfilled.

 

Table1:   EC10-, EC50- (with 95% Confidence Limits) and EC100-Values

(based on the geometric mean measured concentrations of the test item)

Effect levels

 

Test duration

[h]

Tetrapropylenesuccinic acid monoisobutylester

Geometric mean measured test item concentrations

[mg/L]

EC10

(with 95% confidence limits)

24

6.64

(Cl: 5.16 – 8.66)

48

6.22

(Cl: < 2.18 – 11.2)

EC50

(with 95% confidence limits)

24

9.76

(Cl: 8.39 – 11.2)

48

8.03

(Cl: 6.90 – 12.0)

EC100

24

> 13.9

 

48

13.9

 

Categories Display