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 2017-04-18 to 2017-07-17, with the definitive exposure phase from 2017-06-27 to 2017-06-29
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

Test material

Constituent 1
Chemical structure
Reference substance name:
2,4,6-tri-sec-butylphenol
EC Number:
227-572-6
EC Name:
2,4,6-tri-sec-butylphenol
Cas Number:
5892-47-7
Molecular formula:
C18H30O
IUPAC Name:
2,4,6-tris(butan-2-yl)phenol
Test material form:
liquid
Details on test material:
Test item 2,4,6-Tri-sec-Butylphenol
Batch number DEG4355819
CAS No. 5892-47-7
Chemical name 2,4,6-tri-sec-butylphenol
Composition
94.7 [GC area-%] Tri-sec-butylphenol;
2.57 [GC area-%] Di-sec-butylphenol;
1.82 [GC area-%] Tetra-sec-butylphenol
Water solubility 0.79 mg/L
Appearance yellowish slightly viscous clear liquid
Density 0.91 g/cm³ (20 °C)
Stability under test conditions not specified
Expiry date 2017-07-07
Recommended storage tightly closed in a cool, well-ventilated place.

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 fresh media at the start of exposure and at renewal of the test solutions (0 and 24 hours) and in 24-hours old media at renewal and at the end of the exposure (24 and 48 hours). Additionally, the stock solution was analyzed before the start of the exposure phase (0 hours) and before renewal of the test solutions (24 hours).
The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000). Details of the analytical method are presented in section 14. Analytical results are presented in section 7.1.5.

Sampling for the analytical monitoring
At the start of exposure and at renewal of the test solutions (0 and
24 hours), sampling was carried out after preparation of the concentration levels.
At renewal and at the end of exposure (24 and 48 hours), samples of the 24-hours old media were taken from additional replicates prepared with test media, but without daphnids. These additional replicates were incubated under test conditions until analysis.

Criteria for the analytical monitoring
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 stock solution
A stock solution with a nominal loading of 8.79 µL/L of the test item,
which corresponds to a nominal concentration of 8.00 mg test item/L (density of 0.91 g/cm³ taken into account), was prepared one day prior to the start of the exposure (at -24 hours) and one day before the renewal of the test solutions (0 hours) in a glass flask.
The glass flask was filled with an appropriate amount of demineralized water. The test item was introduced into the dilution water and stirred on a magnetic stirrer at approximately 1100 rpm for 24 hours. After completion of the stirring period, the stock solution was allowed to stand for approximately 1 hour and was removed thereafter from the approximate center of the aqueous phase. Thereafter, the mineral components of the dilution water (Elendt M4 medium as specified in Table 2) were added. The stock solution was used as the highest concentration level and for preparation of further concentration levels.
The stock solution prepared at -24 hours was also used for the respective algae study with the test item. For this purpose, an aliquot without the mineral salts was provided.

Test concentrations
7 concentration levels of the test item in a geometric series with a separation factor of 2, prepared by diluting the stock solution with dilution water, was tested as follows:
0.125 - 0.250 - 0.500 - 1.00 - 2.00 – 4.00 – 8.00 mg/L (density of the test item of 0.91 g/cm³ taken into account)
The test item concentrations mentioned above are selected based on the results of the first definitive test and a non-GLP preliminary range finding test (for results, please refer to section 7.1).

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 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. The composition of the culture medium is presented in Table 2.

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

Test conditions

Hardness:
Dilution water
0 hours:Total hardness [mg CaCO3/L]: 174
24 hours:Total hardness [mg CaCO3/L]: 165

Test temperature:
18 - 22°C, constant within ± 1°C
19 °C
pH:
Water Quality Parameters in fresh Media at the Start of the Exposure and at Renewal
(0 and 24 hours)
(measured in one additional replicate (without daphnids) per concentration level and control)
Geometric mean
measured test item
concentration
[mg/L] 0 hours 24 hours
pH-value Dissolved
O2 concentration
[mg/L] pH-value Dissolved
O2 concentration [mg/L]
1.45 8.27 8.30 7.72 8.53
0.759 7.81 8.95 7.68 8.66
0.398 7.70 9.06 7.50 8.67
0.209 7.68 9.09 7.46 8.75
0.112 7.64 9.13 7.48 8.74
0.0407 7.65 9.11 7.50 8.75
0.0106 7.54 9.13 7.92 8.70
Control 7.72 9.22 7.46 9.17


Table 10: Water Quality Parameters in the 24-hours old Media at Renewal and at the End of the Exposure (24 and 48 hours)
(measured in one appropriate replicate (containing daphnids) per concentration level and control)
Geometric mean
measured test item
concentration
[mg/L] 24 hours 48 hours
pH-value Dissolved
O2 concentration
[mg/L] Replicate number pH-value Dissolved
O2 concentration
[mg/L] Replicate number
1.45 7.55 8.67 2 7.46 7.89 4
0.759 7.53 8.59 1 7.47 6.78 1
0.398 7.54 8.70 2 7.47 6.87 4
0.209 7.52 8.68 2 7.45 6.70 2
0.112 7.52 8.65 1 7.46 7.08 1
0.0407 7.49 8.74 1 7.45 7.32 1
0.0106 7.55 8.75 1 7.48 7.46 1
Control 7.60 8.83 1 7.49 8.47 1

Dissolved oxygen:
see above
Conductivity:
Dilution water day 0: 435 [µS/cm]
Dilution water day 1: 423 [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:
EC10
Effect conc.:
1.39 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0.262 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
1.45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.675 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 1.45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
1.45 mg/L
Nominal / measured:
meas. (geom. mean)
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 06-01 to 2017-06-02. For results of the most recent of the monthly performed reference tests, see Table 8.

Table 8: 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 2.05 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 1.80 - 2.33 mg/L


A reference test was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in Elendt M4 medium (Table 2) under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility. The results of the most recent test are presented in section 7.1.7.

Reference item Potassium dichromate p.a. (SIGMA)

Purity 99.0%

Batch number MKBV0900V

Expiry date 2021-11-25

Test concentrations 1.00 – 2.00 – 4.00 mg/L

Ranges of validity EC50 (24 hours): 0.6 mg/L - 2.4 mg/L, according to AQS P 9/2 (clone 5),
EC50 (24 hours): 0.6 mg/L - 2.1 mg/L, according to OECD 202 (clone A)

Exposure phase 2017-06-01 to 2017-06-02

Reported statistics and error estimates:
Methods of evaluation The EC100-values (after 24 and 48 hours) were empirically derived from the observation data (lowest concentration level resulting in 100% immobilization).
All effect concentrations (EC10 / 50 / 100) given are based on the geometric mean measured concentrations, since the measured test item concentrations were not within the range of ± 20% of the nominal concentrations. The concentration-effect relationships of the test item after 24 and 48 hours is shown graphically.

EC-values and statistical The EC10- and the EC50-values after 24 and 48 hours were calculated
statistical analyses 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. In case that no confidence limit were calculated by the software, the highest concentration level resulting in 0% immobilization (EC0) and the lowest concentration level causing 100% immobilization (EC100) were chosen as lower and upper confidence limits.
The EC50-value for the reference item and its confidence limits were calculated accordingly.

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

1.1         Non-GLP Preliminary Range Finding Test

A non-GLP preliminary range finding test under static conditions over a period of 48 hours was conducted at the test facility. Three concentration levels 0.08, 0.8 and 8 mg/L of the test item were prepared with Elendt M4 medium (Table 2) and tested. The preliminary range finding test was conducted under diffuse light conditions (light intensity of max. 1500 lx, 16/8 hours light/dark cycle).

A stock solution of 8 mg/L was prepared one day prior to the start of the exposure intervals (-24 and 0 hours) as specified in section4.1.2andwas also used as highest concentration level. All concentration levels were visually clear throughout the exposure period.

In the range finding test, two replicates per treatment group and control, each with ten daphnids, were tested. The results are presented inTable3andTable4.

 

Table3:      Immobilization Rates in the non-GLP Preliminary Range Finding Test

                (n = 20, divided into 2 replicates with 10 daphnids each)

Nominal

test item

concentration

[mg/L]

IMMOBILIZATION [%]

24 hours

48 hours

Replicates

Replicates

1

2

MV

1

2

MV

8

100

100

100

100

100

100

0.8

   0

   0

   0

 70

 80

 75

0.08

   0

   0

   0

   0

   0

   0

Control

   0

   0

   0

   0

   0

   0

 

Table4:      MeasuredConcentrations of2,4,6-Tri-sec-Butylphenolduring the non-GLPPreliminary Range Finding Test

Sampling date

2016-12-06

0 hours

Start of the

exposure interval

2016-12-07

24 hours

End of the

exposure interval

2016-12-07

24 hours

Start of the

exposure interval

2016-12-08

48 hours

End of the

exposure interval

Nominal

test item

concentration

[mg/L]

2,4,6-Tri-sec-Butylphenol

Meas. conc.

[mg/L]

%

Meas. conc.

[mg/L]

%

Meas. conc.

[mg/L]

%

Meas. conc.

[mg/L]

%

8

8.03

100

2.072)

26

6.36

80

Not determined1)

0.8

0.762

95

0.1852)

23

0.818

102

0.380

48

0.08

0.05202)

65

< LCL2)

0.1082)

136

0.0734

92

Control

< LCL

< LCL

< LCL

< LCL

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

LCL              = lowest calibration level (0.02 mg test item/L)

1)                   = not determined, due to 100% mortality after 24 hours

2)                   = reanalyzed


 

Method of determination          Analytical evaluation of2,4,6-Tri-sec-Butylphenolwas carried out via LC-MS/MSon a reversed phase column in gradient mode.An electrospray tandem mass spectrometer operating in positive ion mode was used as detector. The test item was used as external standard for calibration. For quantification a quadratic calibration curve was used.

 

Equipment                              Autosampler:                           Acquity UPLC,Waters

Binary Solvent Manager:          Acquity UPLC,Waters

Column Manager:                    Acquity UPLC,Waters

                                              Detector:                                 Mass selective detector, Xevo

                                                                                              TQD MS, Acquity UPLC,Waters

Software:                                MassLynxTM4.1,Waters

 

Additional equipment              Piston stroke pipettes, Finnpipette,Thermo scientific

                                              Positive-displacement pipettes,Gilson Medical

                                              Rotary evaporator,Büchi

                                              Ultrasonic bath, Sonorex Super RK 510 H,Bandelin

                                              Strata-X cartridges,Phenomenex

 

Reagents                                HPLC water, for HPLC,VWR

Acetonitrile, super gradient,VWR

Methanol, gradient grade, VWR

 

Standard                                 The test item was used as external standard.

 

Conditions of analysis

 

Column                                   Acquity UPLC BEH C8, 1.7 µm, 50 x 2.1 mm, batch: 0140,Waters

Column temperature                 40°C

Mobile phase (gradient)           A:HPLC water
B:Methanol

 

Table12:    Gradient Table

Time [min]

A [%]

B [%]

0.00

75

25

0.30

75

25

1.00

10

90

2.50

10

90

2.60

75

25

3.00

75

25

 

Flow rate                                 0.5 mL/min

Run time                                 3 min

Injection volume                      10 µL


 

Conditions of detection

 

Detection mode                       Multiple Reaction Mode (MRM)

Ionization mode                       Electrospray negative

Capillary voltage                      2.00 kV

Source temperature                 150 °C

Desolvation gas flow (N2)         1000 L/h

Desolvation temperature          600 °C

Cone voltage                           54 V

Cone Gas Flow                        50 L/h

Parent ion                               261.20 Da

Daughter ion (quantifier)           231.18 Da

Collision energy (quantifier)      30 eV

Collision gas (Ar) pressure       approx. 2.8 x 10-3mbar

Dwell time                               0.155 s

 

Preparation of the standards    A stock solution of 1000 mg test item /L was prepared in acetonitrile and diluted to 10 mg/L with acetonitrile. This solution was further diluted with methanol : HPLC water (50 : 50) to 7 concentrations and used for calibration. For calibration range seeTable 20.

 

Preparation of the                    The method was validated at 0.6 µg test item/L (1x LOQ) and

fortified samples                     and 6.0 µg test item/L (10x LOQ).For the preparation of the fortified samples seeTable13andTable 14.

 

Table13:    Preparation Stepsin Algae Dilution Water

LOQ Level

Control

1

10

Stock solution [mg test item/L]

-

1000

Medium

-

Acetonitrile

Spiking solution

-

60 µg/L (Acetonitrile)

600 µg/L (Acetonitrile)

Replicates

2

5

5

Concentration of the LOQ [µg test item/L]

-

0.6

6

Medium for preparation

Algae dilution water

Volume of spiking solution [mL]

-

1.0

0.1

Volume of medium [mL]

100

100

10

Enrichment factor

100

100

10

Absorption medium [mL]

1.0 (Methanol)

Sample Volume [mL]

0.5

0.5

0.5

Dilution medium [mL]

0.5 (HPLC water)

Finale volume [mL]

1.0

1.0

1.0

Dilution factor

2

2

2

Total enrichment factor

50

50

5

 


 

 

Table14:    Preparation Steps in Daphnia Dilution Water

LOQ Level

Control

1

Control

75

Stock solution [mg test item/L]

-

1000

-

1000

Medium

-

Acetonitrile

-

Acetonitrile

Spiking solution

-

60 µg/L (Acetonitrile)

-

5000 µg/L (Acetonitrile)

Replicates

1

2

1

3

Concentration of the LOQ
[µg test item/L]

-

0.6

-

45

Medium for preparation

daphniadilution water

Volume of spiking solution [mL]

-

1.0

-

0.045

Volume of medium [mL]

100

100

5

5

Enrichment factor

100

100

-

Absorption medium [mL]

1.0 (Methanol)

-

Sample Volume [mL]

0.5

0.5

-

Dilution medium [mL]

0.5 (HPLC water)

5 (Methanol)

Finale volume [mL]

1.0

1.0

10

10

Dilution factor

2

2

2

2

Total enrichment factor

50

50

-

-

 

 


 

Preparation of the samples      The samples and the control were given stabilized for dilution and without stabilization for enrichment steps.
Diluted samples: The samples were stabilized dilution factor 2 with methanol directly after sampling. Further dilutions were made with methanol : HPLC water (50 : 50).
Enriched samples: The first dilution step was made with HPLC water and the second with methanol : HPLC water (50 : 50).
For enrichment and dilution steps seeTable
15toTable18.

 

Table15:    Dilution Steps for0 hours (Start of the first exposure interval)

Nominal

test item

concentration

[mg/L]

Enrichment

factor

 

 

Sample

volume

 

[mL]

Final

volume

 

[mL]

Dilution

factor

 

 

Sample

volume

 

[mL]

Final

volume

 

[mL]

Total factor

8.00

(Stock solution)

1

-

-

80*)

0.125

5.0

Dilution factor 80

8.00

1

-

-

80*)

0.125

5.0

Dilution factor 80

4.00

1

-

-

20*)

0.1

1.0

Dilution factor 20

2.00

1

-

-

10*)

0.2

1.0

Dilution factor 10

1.00

1

-

-

 5*)

0.4

1.0

Dilution factor 5

0.500

1

-

-

 2.5*)

0.8

1.0

Dilution factor 2.5

0.250

1

-

-

 2*)

1.0

1.0

Dilution factor 2

0.125

90

 90

1

16

0.51)

0.1252)

1.0

1.0

Enrichment factor 5.625

Control

100

100

1

 2

0.5

1.0

Enrichment factor 50

*)   = including dilution step (factor 2) directly after sampling

1)   = first dilution step

2)   = second dilution step

 

Table16:    Dilution Steps for24 hours (End of the first exposure interval)

Nominal

test item

concentration

[mg/L]

Enrichment

factor

 

 

Sample

volume

 

[mL]

Final

volume

 

[mL]

Dilution

factor

 

 

Sample

volume

 

[mL]

Final

volume

 

[mL]

Total factor

8.00

1

-

-

20*)

0.1

1.0

Dilution factor 20

4.00

1

-

-

10*)

0.2

1.0

Dilution factor 10

2.00

1

-

-

 5*)

0.4

1.0

Dilution factor 5

1.00

1

-

-

 2.5*)

0.8

1.0

Dilution factor 2.5

0.500

1

-

-

 2*)

1.0

1.0

Dilution factor 2

0.250

100

100

1

20

0.51)

0.12)

1.0

1.0

Enrichment factor 5

0.125

100

100

1

20

0.51)

0.12)

1.0

1.0

Enrichment factor 5

Control

100

100

1

 2

0.5

1.0

Enrichment factor 5

*)   = including dilution step (factor 2) directly after sampling

1)   = first dilution step

2)   = second dilution step


 

 

Table17:       Dilution Steps for24 hours (Start of the second exposure interval)

Nominal

test item

concentration

[mg/L]

Enrichment

factor

 

 

Sample

volume

 

[mL]

Final

volume

 

[mL]

Dilution

factor

 

 

Sample

volume

 

[mL]

Final

volume

 

[mL]

Total factor

8.00

(Stock solution)

1

-

-

80*)

0.125

5.0

Dilution factor 80

8.00

1

-

-

80*)

0.125

5.0

Dilution factor 80

4.00

1

-

-

50*)

0.2

5.0

Dilution factor 50

2.00

1

-

-

25*)

0.08

1.0

Dilution factor 25

1.00

1

-

-

10*)

0.2

1.0

Dilution factor 10

0.500

1

-

-

 5*)

0.4

1.0

Dilution factor 5

0.250

1

-

-

 2*)

1.0

1.0

Dilution factor 2

0.125

1

-

-

 2*)

1.0

1.0

Dilution factor 2

Control

100

100

1

 2

0.5

1.0

Enrichment factor 50

*)   = including dilution step (factor 2) directly after sampling

 

 

Table18:       Dilution Steps for 48 hours (End of the second exposure interval)

Nominal

test item

concentration

[mg/L]

Enrichment

factor

 

 

Sample

volume

 

[mL]

Final

volume

 

[mL]

Dilution

factor

 

 

Sample

volume

 

[mL]

Final

volume

 

[mL]

Total factor

8.00

1

-

-

40*)

0.05

1.0

Dilution factor 40

4.00

1

-

-

20*)

0.1

1.0

Dilution factor 20

2.00

1

-

-

10*)

0.2

1.0

Dilution factor 10

1.00

1

-

-

5*)

0.4

1.0

Dilution factor 5

0.500

1

-

-

2.5*)

0.8

1.0

Dilution factor 2.5

0.250

1

-

-

2*)

1.0

1.0

Dilution factor 2

0.125

100

100

1

20

0.51)

0.12)

1.0

1.0

Enrichment factor 5

Control

100

100

1

2

0.5

1.0

Enrichment factor 50

*)   = including dilution step (factor 2) directly after sampling

1)   = first dilution step

2)   = second dilution step

 

 

Sample storage                       All original samples were storedat room temperature before preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

 

Evaluation                               Quantification of the test item was calculated by peak areabased on the external standard calibration.


 

1.1.1     Method Validation

 

Requirement of the                 According toSANCO 3029/99 rev. 4 (2000)using following criteria.

method validation                    The results inTable19andTable20demonstrate the validity of the analytical method.

 

Table19:    Parameter, Acceptance Criteria and Results of the Method Validation

Parameter

Acceptance criteria

Result

Linearity

 

5 standard concentrations,
r2≥ 0.992

20 to 60 µg test item/L (n = 6),
r2≥ 0.992

ü

Lowest calibration standard

S/N9 for quantifier ion trace

20 µg test item/L, S/N =36

ü

Limit of Detection (LOD)

S/N3 for quantifier ion trace

Not necessary, if the S/N of the lowest calibration standard is > 30

-

Limit of Quantification (LOQ)

At least 20% above lowest calibration level after sample preparation

Algae dilution water:

0.6 µg test item/L (1 x LOQ)
6.0 µg test item/L (10 x LOQ)

Daphniadilution water:

0.6 µg test item/L (1 x LOQ)

45 µg test item/L (75 x LOQ)

ü

Accuracy1)
(Fortified samples)

Mean recovery rate of 70-110% (ideally 80-100%) per
fortification level (2 levels)

Algae dilution water:

1 x LOQ: 78% (n = 5)
10 x LOQ: 88% (n = 5)

Daphniadilution water*):

75 x LOQ: 107% (n = 3)

ü

Precision1)

Relative standard deviation20% per fortification level

Algae dilution water:

1 x LOQ: 2.8%
10 x LOQ: 5.8%

Daphnia dilution water*):

75 x LOQ: 2.6

ü

Specificity
(LC-MS/MS)

Measurement of one specific transition of the precursor ion (used for evaluation). Due to analyte properties no more stable transitions can be observed.

quantifier [m/z]: 261.20 > 231.18.

ü

Blank values < 30% of LOQ

Blank values < 30% of LOQ

ü            = criterion fulfilled

1)             = for results seeTable20

*)            = The most complex medium of all studies of aquatic toxicology was the medium for thealgaestudy (acc. to OECD 201), and was therefore selected exemplarily for the method validation of all study types. This method validation was checked by analysis of replicates prepared in thedaphniadilution water as described above.


 

Table20:    Recovery Rates of the Fortified Samplesof the Test Item2,4,6-Tri-sec-Butylphenol

                                                                       Fortified concentrations*:
0.602 µg test item/L (1 x LOQ), 6.02 µg test item/L (10 x LOQ) and 0.0518 mg test item/L (75 x LOQ)

Replicate

2,4,6-Tri-sec-Butylphenol

Algae dilution water

Daphniadilution water

1 x LOQ

10 x LOQ

1 x LOQ

75 x LOQ

Meas.
conc.
[µg/L]

%

Meas.
conc.
[µg/L]

%

Meas.
conc.
[µg/L]

%

Meas.
conc.
[mg/L]

%

1

0.451

75

7.311)

1211)

0.460

76

0.0558

108

2

0.460

76

4.97

 82

0.421

70

0.0565

109

3

0.480

80

5.57

 92

 

0.0537

104

4

0.476

79

5.54

 92

 

5

0.481

80

5.10

 85

Mean

0.47

78

5.29

 88

0.055

107

SD ±

0.01

 

0.31

 

0.001

 

CV [%]

2.8

 

5.8

 

 2.6

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

%                     = percent of the fortified concentration

*                       = weighing factor taken into account

1)                                  = outlier according to Grubb’s, value was not taken into account for evaluation


2)                                   

Water Quality Parameters in fresh Media at the Start of the Exposure and at Renewal
(0 and 24 hours)

                     (measured in one additional replicate (without daphnids) per concentration level and control)

Geometric mean

measured test item

concentration

[mg/L]

0 hours

24 hours

pH-value

Dissolved
O2concentration
[mg/L]

pH-value

Dissolved
O2concentration [mg/L]

1.45

8.27

8.30

7.72

8.53

0.759

7.81

8.95

7.68

8.66

0.398

7.70

9.06

7.50

8.67

0.209

7.68

9.09

7.46

8.75

0.112

7.64

9.13

7.48

8.74

0.0407

7.65

9.11

7.50

8.75

0.0106

7.54

9.13

7.92

8.70

Control

7.72

9.22

7.46

9.17

 

 

Table10:    Water Quality Parameters in the 24-hours old Media at Renewal and at the End of the Exposure (24 and 48 hours)

                     (measured in one appropriate replicate (containing daphnids) per concentration level and control)

Geometric mean

measured test item

concentration

[mg/L]

24 hours

48 hours

pH-value

Dissolved
O2concentration
[mg/L]

Replicate number

pH-value

Dissolved
O2concentration
[mg/L]

Replicate number

1.45

7.55

8.67

2

7.46

7.89

4

0.759

7.53

8.59

1

7.47

6.78

1

0.398

7.54

8.70

2

7.47

6.87

4

0.209

7.52

8.68

2

7.45

6.70

2

0.112

7.52

8.65

1

7.46

7.08

1

0.0407

7.49

8.74

1

7.45

7.32

1

0.0106

7.55

8.75

1

7.48

7.46

1

Control

7.60

8.83

1

7.49

8.47

1

 

 

Table11:    Water Quality Parameters of the Dilution Waterat the Start of the Exposure and at Renewal
(0 and 24 hours)

Dilution water

dated:

pH-Value

 

 

Dissolved

O2concentration

[mg/L]

Temperature

 

[°C]

Conductivity

 

[µS/cm]

Total hardness

 

[mg CaCO3/L]

0 hours:

2017-06-27

7.72

9.22

20.0

435

174

24 hours:

2017-06-28

7.46

9.17

20.1

423

165

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:

Based on the geometric mean measured concentrations of the test item 2,4,6-Tri-sec-Butylphenol, the 48 hours-EC50 for Daphnia magna was 0.675 mg/L (95% confidence limits: 0.531 – 0.851 mg/L).
Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item2,4,6-Tri-sec-Butylphenol(batch number:DEG4355819) were determined at the test facility according to OECD 202 (2004) from2017‑04-18 to 2017-07-17, with the definitive exposure phase from 2017‑06-27 to 2017-06-29.

The study was conducted under semi-static conditions over a period of 48 hours with seven dilution levels (nominal: 0.125 to 8.00 mg/L) prepared from the stock solution in a geometric series with a separation factor of 2.

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 were analytically verified via LC-MS/MS in fresh media at the start of the exposure and at renewal (0 and 24 hours) and in old media at renewal and at the end of the test (24 and 48 hours) in all concentration levels and the control. Details of the analytical method are presented in section14.

The measured concentrations of the test item in fresh media were in the range of 6 to 26% of the nominal concentrations at the start of the exposure (0 hours) and 28 to 42% at renewal (24 hours). The measured concentrations in corresponding old media were in the range of 3 to 14% of the nominal concentrations at renewal (24 hours) and 6 to 21% of the nominal concentration of the test item at the end of the test (48 hours).The analytical results arepresented inTable7.

All effect concentrations given inTable 1are based on the geometric mean measured concentrations of the test item2,4,6-Tri-sec-Butylphenol.

The validity criteria of the test guideline were fulfilled.

 

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

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

2,4,6-Tri-sec-Butylphenol

Effect concentrations

 

Test duration

[hours]

Geometric mean measured test item concentrations

[mg/L]

EC10

(with confidence limits)

24

  1.39

(CI: 0.757 – > 1.45)

48

  0.262

(CI: 0.112 – 0.418)

EC50

(with confidence limits)

24

  1.45

(CI: 0.757 – > 1.45)

48

  0.675

(CI: 0.531 – 0.851)

EC100

24

> 1.45

 

48

  1.45