Reaction products of alcohols, C11-14-iso, C13 rich and phosphorus pentoxide

Adsorption / desorption

Link to relevant study record(s)

Reference

Endpoint:

adsorption / desorption: screening

Remarks:

Estimation of the Adsorption Coefficient (KOC) on Soil and on Sewage Sludge using High Performance Liquid Chromatography (HPLC)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-17 to 2019-01-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 121 (Estimation of the Adsorption Coefficient (Koc) on Soil and on Sewage Sludge using High Performance Liquid Chromatography (HPLC))

Version / remarks:

acc. to OECD 121 (2001) and Council Regulation (EC) No. 440/2008, C.19

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of method:

HPLC estimation method

Media:

soil/sewage sludge

Radiolabelling:

no

Test temperature:

20 - 25 ± 1 °C.

Details on study design: HPLC method:

Method

Test System Analytical HPLC-column (based on silica matrix), coupled to DAD and MS, packed with a cyanopropyl solid phase containing non-polar and polar moieties. Methanol : ammonium formate buffer pH 5.5 (50 : 50 v/v) and methanol : ammonium formate buffer pH 7.5 (50 : 50 v/v) were used as mobile phases.
 
Reasons for the selection The selection of the test system is based on the guidelines. HPLC coupled to DAD and MS is a useful method for estimation of the adsorption coefficient KOC. The KOC indicates the binding capacity of the test item on soil or sewage sludge and allows comparing the adsorption behaviour of different chemicals. The test item was detected by MS and the reference items were detected by DAD.

Analytical system:
Autosampler: Acquity UPLC, WATERS
Binary Solvent Manager: Acquity UPLC, WATERS
Column Manager: Acquity UPLC, WATERS
DAD-Detector: Acquity UPLC Photodiode Array Detector, WATERS
MS-Detector: Mass selective detector, TQD, Acquity UPLC, WATERS
Software: Empower 2 (Build No: 2154), WATERS

Column:
YMC-Pack Cyano, S-5 µm / 12nm / 250 x 4.0 mm, batch 13303, YMC EUROPE GMBH

Mobile phase:
pH 5.5: Methanol : ammonium formate buffer 10 mM (50 : 50 v/v)
pH 7.5: Methanol : ammonium formate buffer 10 mM (50 : 50 v/v)
Methanol and ultrapure water in a ratio 55 : 45 (system suitability)

Additional equipment:
Analytical balances, SARTORIUS
Data logger/thermometer TESTO 175-T3
pH-Meter, Lab 850, SI ANALYTICS
Positive displacement pipettes, Microman, GILSON
Piston stroke pipettes, Pipetman, GILSON
Ultrapure water generator, MERCK
Thermostat, ecoline RE201, LAUDA
Top pan balance, KERN
Laboratory glassware

Reagents:
3-Nitrobenzamide, 98%, batch 53297LJ, SIGMA-ALDRICH
Ammonium formate, for mass spectroscopy,
batch: BCBX5107, MERCK
Ammonium hydroxide, 25% (w/w) for LC-MS,
batch K50660003 846, MERCK
Endosulfan-diol, 99.9%, batch SZBD302XV, SIGMA-ALDRICH
Formic acid, ≥98%, batch 17E154003, VWR
Methanol, ≥99.9%, batch STBH8356, MERCK
Ultrapure water, produced internally

Column temperature:
The column temperature during the study was monitored every 2 min. by a data logger. The temperature was kept in the required range of 20 - 25 ± 1 °C. Details are given below.

Temperature Range
Study System suitability
Max. Temperature 24.8 °C 25.0 °C
Min. Temperature 24.6 °C 24.8 °C
Mean Value 24.7 °C 24.9 °C

Flow rate:
0.7 mL/min

Injection volume:
25 µL for the test item at pH 5,5
75 µL for the test item at pH 7,5
10 µL for reference items

System suitability:
The following capacity factors k’ were reached and confirmed system suitability:
log k’ > 0 (0.501 measured) for log KOC = 3.02 (Endosulfan-diol) and log k’ > -0.4 (0.119 measured) for log KOC = 1.95 (3-Nitrobenzamide) by using methanol : ultrapure water (55 : 45) as mobile phase.

TEST ITEM:
Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide

Stock solution:
2 g/L in methanol

Test concentration:
100 mg/L for the test item at pH 5,5
10 mg/L for the test item at pH 7,5

Dilution:
With methanol and buffer solution to mobile phase ratio
pH 5.5: Methanol : ammonium formate buffer 10 mM (50 : 50 v/v)
pH 7.5: Methanol : ammonium formate buffer 10 mM (50 : 50 v/v)
The pH values were determined for both test solutions.

Replicates:
Single (three injections)

Detection:
With MS/MS:
Detection mode Multiple Reaction Mode (MRM)
Ionization mode Electrospray positive
Capillary voltage 4.00 kV
Source temperature 150 °C
Desolvation gas flow (N2) 1000 L/h
Desolvation temperature 500 °C
Cone Gas Flow 150 L/h
Dwell time 0.1 s

MS Parameter Test Item:

MRM Channel Parent Ion [Da] Daughter Ion [Da] Cone Voltage [V] Collision Energy [eV]
1 281.23 98.96 58 41
2 281.23 71.08 58 30
3 281.23 57.09 58 30

REFERENCE ITEMS:
Sodium nitrate was used for determination of the dead time of the test system. Uracil was used for determination of the difference of retention times of measurements between DAD and MS. Seven reference items were used for soil and sewage sludge. Six reference items were used for evaluation because aniline was considered unsuitable for both matrices as shown by outlier test (F-Test).

Stock solutions:
100 mg/L in ultrapure water for sodium nitrate and uracil
100 mg/L for the others in methanol

Test concentrations:
20 mg/L for Endosulfan-diol, 5 mg/L for the others

Dilution:
With methanol and buffer solution to mobile phase ratio
pH 5.5: Methanol : ammonium formate buffer 10 mM (50 : 50 v/v)
pH 7.5: Methanol : ammonium formate buffer 10 mM (50 : 50 v/v)

Replicates:
Sodium nitrate (three injections)
Reference items, single with one injection before and one after the test item

Detection:
With DAD and MS
DAD range: 200 – 400 nm (210 – 400 nm for system suitability)

MS/MS for detection of Uracil:

Detection mode Multiple Reaction Mode (MRM)
Ionization mode Electrospray positive
Capillary voltage 3.00 kV
Source temperature 120 °C
Desolvation gas flow (N2) 1000 L/h
Desolvation temperature 500 °C
Cone Gas Flow 100 L/h
Dwell time 0.1 s
Cone Voltage 46 V
Collision Energy 15 V
Mass transition 112.96 > 96.04

The following table shows the run time and the wavelengths used for evaluation.

Reference Item Wavelength [nm] Runtime [min]
Sodium nitrate 202 10
Uracil 250 10
Acetanilide 235 10
Atrazine 220 10
Monuron 250 10
Aniline 235 10
Linuron 250 10
Phenyl benzoate 235 15
Fenthion 250 20
Sodium nitrate* 211 5
Endosulfan-diol* 212 10
3-Nitrobenzamide* 258 10

* for system suitability

Type and frequency of measurement:
Analyses of the test item was carried out with a cyanopropyl analytical column coupled to DAD and MS. The test item and the reference items were dissolved in methanol, diluted to mobile phase ratio and injected into the HPLC system, where the items interacted with the stationary phase while passing through the column. The column temperature was monitored with a thermometer / data logger during the test in 2 minutes intervals. The retention times were determined.

Analytical monitoring:

yes

Type:

log Koc

Value:

1.9 dimensionless

pH:

5.5

Details on results (HPLC method):

At pH 5.5 the logKOC values were determined to be 1.9 for soil and 1.8 for sewage sludge. At pH 7.5 the test item eluted slightly before the reference item with the lowest logKOC value (acetanilide). Therefore, the logKOC value is extrapolated and values of 1.3 for soil and 1.1 for sewage sludge were calculated.
 

Results

 

pH values of the test item in mobile phase

 

The pH values were

5.643 in pH 5.5:Methanol : ammonium formate buffer 10 mM(50 : 50 v/v)

7.005 in pH 7.5: Methanol : ammonium formate buffer 10 mM(50 : 50 v/v)

 

Dead Time of the HPLC-System

HPLC dead time t₀was determined by threefold injection of a solution of 5 mg/L sodium nitrate.

Dead Time of the HPLC-System

Serial No.	Determination	Determination
	t0 for pH 5.5 [min]	t0 for pH 7.5 [min]
1	2.530	2.313
2	2.526	2.314
3	2.528	2.314
Mean ± SD	2.528±0.002	2.314±0.001

            SD = Standard deviation

 

Determination of the Retention Time Difference

 

The retention time difference between DAD and MS was determined by threefold injection of Uracil.

 

Retention Time Difference (dt)

Item	Serial no.	tR, DAD[min]	tmean, DAD[min]	tR, MS[min]	tmean, MS[min]	dt [min]
Uracil pH 5.5	1	3.122	3.121	3.143	3.161	0.040
	2	3.119		3.185
	3	3.123		3.156
Uracil pH 7.5	1	3.133	3.130	3.163	3.165	0.036
	2	3.130		3.187
	3	3.126		3.146

 

 

Calibration Data

 

Retention time data of the reference items used for calibration of soil and sewage sludge are given in Table 1 and Table 3.

The corresponding calibration data for the corresponding calibration lines are summarized in Table 2 and 4. The calibration data were obtained from the regression line of the graph logk’ vs. logK_OC.

 

Table1:      Capacity Factors (k’) of the Reference Items for Soil and Sewage Sludge at pH 5.5

Reference Item	logKOC.soil	logKOC.sewage sludge	1^stCalibration			2^ndCalibration
			tR1 [min]	k'	logk'	tR2 [min]	k'	logk'
Acetanilide	1.26	1.52	3.888	0.54	-0.2692	3.890	0.54	-0.2686
Atrazine	1.81	1.66	4.544	0.80	-0.0983	4.553	0.80	-0.0964
Monuron	1.99	1.46*	4.693	0.86	-0.0673	4.696	0.86	-0.0667
Aniline*	2.07	1.74	3.807	0.51	-0.2959	3.806	0.51	-0.2962
Linuron	2.59	2.43	6.811	1.69	0.2290	6.819	1.70	0.2298
Phenyl benzoate	2.87	3.26	7.097	1.81	0.2570	7.096	1.81	0.2569
Fenthion	3.31	3.75	11.101	3.39	0.5304	11.114	3.40	0.5310

t_R1       = Retention time 1. injection;                         t_R2     = Retention time 2. injection
k’      = Capacity factor;                                           K_OC   = Adsorption coefficient

*)      = The reference item was not used for the evaluation, outlier

 

Table2:      Calibration Data of the Reference Items for Soil and Sewage Sludge at pH 5.5

	Soil		Sewage Sludge
	1^stCalibration	2^ndCalibration	1^stCalibration	2^ndCalibration
slope	2.545	2.547	3.134	3.135
intercept	2.058	2.056	2.043	2.040
r^2	0.9787	0.9785	0.8890	0.8884
r	0.9893	0.9892	0.9429	0.9425

r       = Correlation Coefficient                                r²            = Coefficient

 

Table3:    Capacity Factors (k’) of the Reference Items for Soil and Sewage Sludge at pH 7.5

Reference Item	logKOC.soil	logKOC.sewage sludge	1^stCalibration			2^ndCalibration
			tR1 [min]	k'	logk'	tR2 [min]	k'	logk'
Acetanilide	1.26	1.52	3.890	0.68	-0.1667	3.884	0.68	-0.1683
Atrazine	1.81	1.66	4.531	0.96	-0.0185	4.520	0.95	-0.0206
Monuron	1.99	1.46*	4.701	1.03	0.0136	4.686	1.03	0.0109
Aniline*	2.07	1.74	3.807	0.65	-0.1901	3.796	0.64	-0.1934
Linuron	2.59	2.43	6.804	1.94	0.2880	6.795	1.94	0.2871
Phenyl benzoate	2.87	3.26	7.110	2.07	0.3166	7.103	2.07	0.3160
Fenthion	3.31	3.75	11.053	3.78	0.5772	11.091	3.79	0.5791

t_R1       = Retention time 1. injection;                         t_R2     = Retention time 2. injection
k’      = Capacity factor;                                            K_OC   = Adsorption coefficient

*)      = The reference item was not used for the evaluation, outlier

 

Table4:    Calibration Data of the Reference Items for Soil and Sewage Sludge at pH 7.5

	Soil		Sewage Sludge
	1^stCalibration	2^ndCalibration	1^stCalibration	2^ndCalibration
slope	2.723	2.708	3.368	3.353
intercept	1.846	1.852	1.780	1.786
r^2	0.9759	0.9752	0.8943	0.8953
r	0.9879	0.9875	0.9457	0.9462

r       = Correlation Coefficient;                                r²            = Coefficient

 

Results of Test Item Analysis

The following tables contain the retention time data and calculated logk’ values at pH 5.5 and pH 7.5. Two individual logK_OC values were derived under consideration of the first and second calibration. Finally, a mean value was calculated from these individual logK_OC value for soil and for sewage sludge. At pH 5.5 the logK_OC values were 1.9 for soil and 1.8 for sewage sludge. At pH 7.5 the test item eluted slightly before the reference item with the lowest logK_OC value (acetanilide). Therefore, the logK_OC value is extrapolated and values of 1.3 for soil and 1.1 for sewage sludge were calculated.

Retention Times, Capacity Factors (k’) and Adsorption Coefficient (logK_OC) of
Reaction products of alcohols, C11-14-iso, C13 rich and phosphorus pentoxide for Soil and Sewage Sludgeat pH 5.5

Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxideat pH 5.5
MRM Channel	tR* [min]	k’	logk’	logKOC.soil± SD 95 % CI		logKOC.sewagesludge± SD 95 % CI
				1^stCalibration	2^ndCalibration	1^stCalibration	2^ndCalibration
2	4.724	0.85	-0.0691	1.89 ± 0.06 1.73 to 2.06	1.89 ± 0.06 1.73 to 2.06	1.84 ± 0.17 1.36 to 2.32	1.84 ± 0.17 1.36 to 2.32
1	4.726	0.85	-0.0687
3	4.738	0.86	-0.0663
3	4.728	0.85	-0.0683
2	4.773	0.87	-0.0594
1	4.782	0.88	-0.0576
3	4.688	0.84	-0.0764
2	4.736	0.86	-0.0667
1	4.834	0.90	-0.0475
Mean	4.75	0.86	-0.0645	1.9		1.8

 

K_OC         : Adsorption coefficientaccording to the guidelines

CI           : Confidence Interval

*)            : corrected t_R= measured retention time – retention time shift

 

 

Retention Times, Capacity Factors (k’) and Adsorption Coefficient (logK_OC) of
Reaction products of alcohols, C11-14-iso, C13 rich and phosphorus pentoxide for Soil and Sewage Sludge at pH 7.5

Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxideat pH 7.5 MRM Channel tR [min] k’ logk’ logKOC.soil± SD 95 % CI logKOC.sewagesludge± SD 95 % CI 1^stCalibration 2^ndCalibration 1^stCalibration 2^ndCalibration 3 3.773 0.62 -0.211 1.33 ± 0.09 1.07 to 1.59 1.33 ± 0.09 1.07 to 1.60 1.14 ± 0.25 0.43 to 1.84 1.15 ± 0.25 0.45 to 1.84 2 3.987 0.71 -0.150 3 3.814 0.63 -0.199 2 3.841 0.64 -0.191 3 3.749 0.60 -0.218 2 3.887 0.66 -0.177 Mean 3.84 0.65 -0.191 1.3 1.1

 

K_OC         : Adsorption coefficientaccording to the guidelines

CI           : Confidence Interval

*)            : corrected t_R= measured retention time – retention time shift

 

 

Validity criteria fulfilled:

yes

Executive summary:

Summary

 

The adsorption coefficient (K_OC) for the test item Reaction products of alcohols, C11-14-iso, C13 rich and phosphorus pentoxide (batch number: DEG4410913) on soil and on sewage sludge using high performance liquid chromatography (HPLC) was estimated according to OECD 121 (2001) and Council Regulation (EC) No. 440/2008, Method C.19 from 2019-01-17 to 2019-01-19 at Noack Laboratorien GmbH, 31157 Sarstedt, Germany.

 

Calibrations were performed using six reference items for soil and sewage sludge with known log K_OC values (given by the guidelines). The reference items were measured twice, once before and once after the measurement (double injection of one replicate) of the test item and were detected by DAD. The test item was detected by MS. Sodium nitrate was used for the determination of the dead time of the HPLC system coupled to DAD. Uracil was detected by DAD as well as by MS to derive the retention time shift between DAD and MS. The compounds pass first the DAD and then the MS leading to a higher retention time for the same compound when detected by MS in contrast to detection by DAD. For this reason, the retention time shift was used as correction factor to reduce the retention time of the test item because sodium nitrate as dead time marker was only detected with DAD.

 

At pH 5.5 the log K_OC values were determined to be 1.9 for soil and 1.8 for sewage sludge see the table below. At pH 7.5 the test item eluted slightly before the reference item with the lowest log K_OC value (acetanilide). Therefore, the log K_OC value is extrapolated and values of 1.3 for soil and 1.1 for sewage sludge were calculated (see the second table below).

 

Values of logK_OC of Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide at pH 5.5

Test Item	logKOC, soil± SD 95 % CI		logKOC, sewage sludge± SD 95 % CI
	1. Calibration	2. Calibration	1. Calibration	2. Calibration
	1.89 ± 0.06 1.73 to 2.06	1.89 ± 0.06 1.73 to 2.06	1.84 ± 0.17 1.36 to 2.32	1.84 ± 0.17 1.36 to 2.32
Mean	1.9		1.8

K_OC         : Adsorption coefficientaccording to the guidelines

CI           : Confidence Interval

 

Values of logK_OC of Reaction products of alcohols, C11-14-iso, C13 rich and phosphorus pentoxideat pH 7.5

Test Item	logKOC, soil± SD 95 % CI		logKOC, sewage sludge± SD 95 % CI
	1. Calibration	2. Calibration	1. Calibration	2. Calibration
	1.33 ± 0.09 1.07 to 1.59	1.33 ± 0.09 1.07 to 1.60	1.14 ± 0.25 0.43 to 1.84	1.15 ± 0.25 0.45 to 1.84
Mean	1.3		1.1

K_OC         : Adsorption coefficientaccording to the guidelines

CI           : Confidence Interval

 

The adsorption coefficient logKOC for Reaction products of alcohols, C11-14-iso, C13 rich and phosphorus pentoxideat pH 5.5 was determined to be 1.9 for soil and 1.8 for sewage sludge. The adsorption coefficient logKOC at pH 7.5 was estimated to be 1.3 for soil and 1.1 for sewage sludge.

 

Description of key information

At pH 5.5 the logK_OCvalues were determined to be 1.9 for soil and 1.8 for sewage sludge see the table below. At pH 7.5 the test item eluted slightly before the reference item with the lowest logK_OCvalue (acetanilide). Therefore, the logK_OCvalue is extrapolated and values of 1.3 for soil and 1.1 for sewage sludge were calculated

Key value for chemical safety assessment

Koc at 20 °C:

79.44