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Reference
Endpoint:
adsorption / desorption: screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A modern study conducted in compliance with GLP standards and followed OECD Guidelines
Qualifier:
according to
Guideline:
OECD Guideline 121 (Estimation of the Adsorption Coefficient (Koc) on Soil and on Sewage Sludge using High Performance Liquid Chromatography (HPLC))
Deviations:
no
GLP compliance:
yes
Type of method:
HPLC estimation method
Media:
other: Agilent Poroshell 120 EC-CN (50 x 3.0 mm, 2.7 µm particle size).
Specific details on test material used for the study:
Identifier: EXP1200078
Appearance: Very dark brown (almost black) viscous liquid
Batch: E00275-350
Sample Expiration Date: end-2013
Purity:100%
Radiolabelling:
no
Test temperature:
The column temperature was maintained at 40°C ± 0.8°C.
Details on study design: HPLC method:
Retention times of the test substance and reference substances were determined using an Agilent Series 1100/1200 High Performance Liquid Chromatograph (HPLC) equipped with an Agilent Series 1100 variable wavelength UV detector and an Alltech 500 ELSD. A mean detector offset between the UV and ELSD detectors was 0.214 ± 0.006 minutes (N = 3, RSD = 2.8%). Chromatographic separations were achieved using an Agilent Poroshell 120 EC-CN column (50 x 3.0 mm, 2.7 µm particle size). The column temperature was maintained at 40°C ± 0.8°C. The flow rate was 0.300 mL/minute. The column dead time was determined by injections of the thiourea calibration reference standard solution prior to and following analysis of the test substance.

The six calibration reference standard solutions (10.0 µL injection volume) were injected prior to and after the test substance to determine retention times over the total run time. The test substance solutions were each injected following the initial injections of the reference standard solutions and were analyzed under the same chromatographic conditions as the reference standard solutions.
Analytical monitoring:
no
Details on test conditions:
EQUIPMENT
- Apparatus: Agilent Series 1100/1200 High Performance Liquid Chromatograph (HPLC).
- Type, Agilent Poroshell 120 EC-CN (50 x 3.0 mm, 2.7 µm particle size)

- Detection system:
1. Agilent Series 1100 Variable Wavelength (UV) Detector.
- UV wavelength: 220 nm
2. Alltech 500 Evaporative Light Scattering Detector (ELSD)
- ELSD Parameters:
- Nebulizer Gas = Nitrogen
- Drift Tube Temperature = 87°C
- Gas Flow = ~2.38 SLPM
- ATTN = 1/4
- Gas L = 1.8

MOBILE PHASES
- Solvent A (100%): 55% (1:1 ACN: THF): 45% (H2O)
- Solvent B (0.0%): THF
- Experiments with additives carried out on separate columns: No.
- pH: ~ 6 using using pH indicator paper (Whatman/Lot No. 10B357).
- Solutes for dissolving test and reference substances: Tetrahydrofuran.

DETERMINATION OF DEAD TIME
- Method: Capacity factors were calculated for the reference standards using thiourea to estimate column dead time (i.e. the retention time of an unretained organic compound).

REFERENCE SUBSTANCES
- Identity: Thiourea; Acetanilide; Phenol; Naphthalene; Phenanthrene; DDT.

DETERMINATION OF RETENTION TIMES
- Retention times of the test substance and reference substances were determined using an Agilent Series 1100/1200 High
Performance Liquid Chromatograph (HPLC) equipped with an Agilent Series 1100 variable wavelength UV detector and an
Alltech 500 ELSD. A mean detector offset between the UV and ELSD detectors was 0.214 ± 0.006 minutes (N = 3, RSD = 2.8%). Chromatographic separations were achieved using an Agilent Poroshell 120 EC-CN column (50 x 3.0 mm, 2.7 µm particle
size). The column temperature was maintained at 40°C ± 0.8°C. The flow rate was 0.300 mL/minute. The column dead time was determined by injections of the thiourea calibration reference standard solution prior to and following analysis of the
test substance.

- Since the first two of the six test substance peaks on the UV detector eluted at retention times of approximately 0.83 and
1.06 minutes, respectively, which was prior to the retention time for thiourea, the corresponding log KOC for each peak
could not be extrapolated. Therefore, the log KOC of these peaks will be reported as less than thiourea, an estimated log
KOC of 0.85. Since the fifth UV and first two ELSD peaks eluted during the isocratic portion of the analytical method, but eluted after the first calibration reference standard, DDT (log KOC = 5.63), both an extrapolated and a limit value were
reported for these peaks as per the OECD 121 guideline. Extrapolation can result in uncertainty because assumptions are
made on how the regression trendline will behave outside of the range of the known set of values. The farther the
extrapolated value from the beginning or end of the trendline the greater the uncertainty in the extrapolated value. Since
the sixth UV and third ELSD peaks of the test substance eluted after the last calibration reference standard, DDT, and had to be driven off the column by way of a stronger (i.e. less polar) mobile phase than used for the elution of the reference
substances, the log KOC for the sixth UV and third ELSD peaks of the test substance cannot be extrapolated and were reported as a limit value of greater than 5.63.

REPETITIONS
- Number of determinations: The test substance was injected in triplicate.

Computational methods:
EVALUATION
- Calculation of capacity factors k': calculated using the equation k' = (tR - t0)/t0 where tR was the retention time and t0 was the column dead time.

- Determination of the log Koc value: A correlation graph of log k' versus log Koc for the reference standards was plotted and fitted to a regression equation in the form of y = mx + b. Log Koc for the test substance was calculated by substituting the
calculated logarithm of the capacity factor for the test substance into the linear regression equation for the calibration
curve.
Key result
Sample No.:
#1
Type:
log Koc
Value:
< 0.85 dimensionless
pH:
6
Temp.:
40 °C
Matrix:
Agilent Poroshell 120 EC-CN column
Remarks on result:
other: Peak eluted faster than the thioacetamide peak used to measure 'dead time/volume.'
Key result
Sample No.:
#2
Type:
log Koc
Value:
< 0.85 dimensionless
pH:
4
Temp.:
40 °C
Matrix:
Agilent Poroshell 120 EC-CN column
Remarks on result:
other: Peak eluted faster than the thioacetamide peak used to measure 'dead time/volume.'
Key result
Sample No.:
#3
Type:
log Koc
Value:
1.24 dimensionless
pH:
6
Temp.:
40 °C
Matrix:
Agilent Poroshell 120 EC-CN column
Key result
Sample No.:
#4
Type:
Koc
Value:
4.37 dimensionless
pH:
6
Temp.:
40
Matrix:
Agilent Poroshell 120 EC-CN column
Key result
Sample No.:
#5
Type:
log Koc
Value:
> 5.63 dimensionless
pH:
6
Temp.:
40
Matrix:
Agilent Poroshell 120 EC-CN column
Remarks on result:
other:
Remarks:
Peak elution was longer than DDT which was the standard with the highest Koc used in this study.
Details on results (HPLC method):
Injections of a reagent blank solution (55% 1:1 ACN: THF and 45% H2O (v/v)) showed that the reagents used for preparation of the calibration reference standard solutions were free of any potentially interfering contaminants.

A set of six reference standard solutions were prepared and injected in duplicate (once near the beginning and once near the end of the HPLC sequence). The retention times for one of the reference substances, thiourea, were used to determine the analytical column dead time (t0) for use in calculating capacity factors (k') of the remaining reference substances and the test substance. The mean retention time of thiourea was 1.096 minutes on the UV detector. Five additional reference substances were analyzed. The capacity factors of all the reference substances were calculated based upon their retention times.

The three 1.02 mg/mL test substance solutions were sequentially injected. The test substance eluted on the UV detector as six peaks in each test substance sample. The test substance eluted as three peaks on the ELSD detector. Prior to the injections of the test substance solutions, a THF solvent blank was injected to identify any solvent background peaks. Two peaks at approximately 1.04 and 1.25 minutes, respectively, were present in both the THF solvent blank and test substance chromatograms under UV conditions at 220 nm. In the test substance chromatograms these peaks were significantly larger. Therefore, these peaks are suspected to be components of the test substance.

Since the first two of the six test substance peaks on the UV detector eluted at retention times of approximately 0.83 and 1.06 minutes, respectively, which was prior to the retention time for thiourea, the corresponding log KOC for each peak
could not be extrapolated. Therefore, the log KOC of these peaks will be reported as less than thiourea, an estimated log
KOC of 0.85. Since the fifth UV and first two ELSD peaks eluted during the isocratic portion of the analytical method,
but eluted after the first calibration reference standard, DDT (log KOC = 5.63), both an extrapolated and a limit value
were reported for these peaks as per the OECD 121 guideline. Extrapolation can result in uncertainty because assumptions are made on how the regression trendline will behave outside of the range of the known set of values. The farther the
extrapolated value from the beginning or end of the trendline the greater the uncertainty in the extrapolated value. Since the sixth UV and third ELSD peaks of the test substance eluted after the last calibration reference standard, DDT, and
had to be driven off the column by way of a stronger (i.e. less polar) mobile phase than used for the elution of the
reference substances, the log KOC for the sixth UV and third ELSD peaks of the test substance cannot be extrapolated and were reported as a limit value of greater than 5.63.

The capacity factors (k') of each test substance peak on the UV and ELSD were then calculated based on the corresponding retention time. The corresponding mean log KOC for each of the six UV test substance peaks was calculated as < 0.85, < 0.85, 1.24, 4.37, > 5.63 (6.01 extrapolated) and > 5.63, respectively. The corresponding mean log KOC for each of the
three ELSD test substance peaks was calculated as > 5.63 (6.00 extrapolated), > 5.63 (8.07 extrapolated) and > 5.63,
respectively.
Transformation products:
not measured
Remarks:
This material is described as an UVCB substance, and individual components could not be identified or quantitated even with the use of radiolabeling.

Item Content
Table 1 HPLC Operational Parameters
Table 3 Calibration Reference Standards Retention Times, Capacity Factors, and Log KOC Values
Table 4 Log Adsorption Coefficients (Log KOC) Based on UV Data for the Test Substance
Table 5 Log Adsorption Coefficients (Log KOC) Based on ELSD Data for the Test Substance
   
Figure 1 Reference standard calibration curve
Validity criteria fulfilled:
yes
Conclusions:
The capacity factors (k') of each test substance peak on the UV and ELSD were then calculated based on the corresponding retention time. The corresponding mean log KOC for each of the six UV test substance peaks was calculated as < 0.85, < 0.85, 1.24, 4.37, > 5.63 (6.01 extrapolated) and > 5.63, respectively. The corresponding mean log KOC for each of the three ELSD test substance peaks was calculated as > 5.63 (6.00 extrapolated), > 5.63 (8.07 extrapolated) and > 5.63, respectively.
Executive summary:

Estimation of absorption coefficients (Koc) of EXP1200078 was conducted by HPLC using a set of six stands and diluent. The study was done following OECD Guideline 121.

Under the chromatographic conditions specified, the test substance eluted as six peaks on the Ultraviolet

(UV) detector and three peaks on the Evaporative Light Scattering Detector (ELSD). The log KOCof the test substance was a range of < 0.85 to > 5.63.

Description of key information

Under the chromatographic conditions specified, the test substance eluted as six peaks on the Ultraviolet (UV) detector and three peaks on the Evaporative Light Scattering Detector (ELSD). The log KOCof the test substance was a range of < 0.85 to > 5.63.

Key value for chemical safety assessment

Koc at 20 °C:
53 703

Additional information

[LogKoc: 4.73]