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EC number: 227-572-6 | CAS number: 5892-47-7
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 2 017
- 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
- 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.
Constituent 1
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 µEm-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 |
- |
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 seeTable15toTable18.
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, |
20 to 60 µg test item/L (n = 6), |
ü |
||||
Lowest calibration standard |
S/N≥9 for quantifier ion trace |
20 µg test item/L, S/N =36 |
ü |
||||
Limit of Detection (LOD) |
S/N≥3 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) Daphniadilution water: 0.6 µg test item/L (1 x LOQ) 45 µg test item/L (75 x LOQ) |
ü |
||||
Accuracy1) |
Mean recovery rate of 70-110% (ideally 80-100%) per |
Algae dilution water: 1 x LOQ: 78% (n = 5) Daphniadilution water*): 75 x LOQ: 107% (n = 3) |
ü |
||||
Precision1) |
Relative standard deviation≤20% per fortification level |
Algae dilution water: 1 x LOQ: 2.8% Daphnia dilution water*): 75 x LOQ: 2.6 |
ü |
||||
Specificity |
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. |
% |
Meas. |
% |
Meas. |
% |
Meas. |
% |
||||||||||
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 |
pH-value |
Dissolved |
|
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 |
Replicate number |
pH-value |
Dissolved |
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
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