2-[N-(2-cyanoethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]anilino]ethyl acetate

Adsorption / desorption

Link to relevant study record(s)

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:

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))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.19 (Estimation of the Adsorption Coefficient (KOC) on Soil and Sewage Sludge Using High Performance Liquid Chromatography (HPLC))

GLP compliance:

yes

Type of method:

HPLC estimation method

Media:

soil/sewage sludge

Radiolabelling:

no

Test temperature:

The column temperature during the study was monitored every 2 min by a data logger. A mean value of 21.8°C was obtained for the temperature during the course of the study.

Details on study design: HPLC method:

TEST SYSTEM Analytical HPLC-column (based on silica matrix), coupled to DAD, packed with a cyanopropyl solid phase containing non-polar and polar moieties. Methanol and HPLC water in a ratio of 70:30 was used as mobile phase.


Reasons for the selection The selection of the test system is based on the guidelines. HPLC coupled to DAD 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 and 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
Software: Empower 2 (Build No: 2154), WATERS

Column YMC-Pack Cyano / S-5 µm / 12 nm, 250 x 4.0 mm, batch: 13303, YMC EUROPE GMBH 
Mobile phase Methanol : HPLC water (70:30 v/v)

Additional equipment Data logger/thermometer TESTO 175-T3
Analytical balances, SARTORIUS
Top pan balance, KERN
Positive displacement pipettes, Microman, GILSON
Piston stroke pipettes, Finnpipette, THERMO SCIENTIFIC
Laboratory glassware

Reagents HPLC water, for HPLC, batch 16H184002, VWR
Methanol, gradient grade, batch 16G224004, VWR
3-Nitrobenzamide, 98%, batch 53297LJ, ALDRICH
Endosulfan-diol, 99.9%, batch SZBD302XV, SIGMA-ALDRICH

Column temperature 20 - 25 ± 1°C

Flow rate 0.7 mL/min

Injection volume 10 µL

Temperature monitoring Data logger using 2 min interval

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

Analytical monitoring:

not required

Details on sampling:

TEST ITEM Disperse Orange 30

Stock solution 100 mg/L in methanol

Test concentration 10 mg/L

Dilution With methanol and HPLC water to mobile phase ratio

Replicates Single (three injections)

Detection With DAD

Wavelength Detected range: 210 nm – 500 nm
Data evaluated at 416 nm



REFERENCE ITEMS NaNO3 was used for determination of dead time of the test system. Eight reference items were analysed as follows.

Stock solutions 100 mg/L in HPLC water for NaNO3
100 mg/L for the others in methanol

Test concentration 5 mg/L

Dilution With methanol and HPLC water to mobile phase ratio

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

Key result

Type:

log Koc

Value:

4.36 dimensionless

Matrix:

Soil

Remarks on result:

other: peak 1

Key result

Type:

log Koc

Value:

4.43 dimensionless

Matrix:

Sewage sludge

Remarks on result:

other: peak 1

Key result

Type:

log Koc

Value:

5.13 dimensionless

Matrix:

Soil

Remarks on result:

other: peak 2

Key result

Type:

log Koc

Value:

5.31 dimensionless

Matrix:

Sewage sludge

Remarks on result:

other: peak 2

Details on results (HPLC method):

Temperature

The column temperature during the study was monitored every 2 min. by a data logger. Details are given in the table below.

Temperature Range
Min. Temperature 20.7°C
Max. Temperature 22.9°C
Mean Value 21.8°C


Dead Time of the HPLC-System
HPLC dead time t0 was determined by threefold injection of a solution of 5 mg/L sodium nitrate (table below).

Dead Time of the HPLC-System
Serial No. Determination at pH 5.5
t0 [min]
1 1.930
2 1.931
3 1.931
Mean ± SD 1.931 +/- 0.0006
SD = Standard deviation

6.3 Calibration Data

Retention time data of the reference items used for calibration of soil and sewage sludge are given in table below.

Capacity Factors (k’) of the Reference Items for Soil and Sewage Sludge
1st Calibration 2nd Calibration
Reference Item log KOC. soil log KOC. sewage sludge tR1 [min] k' log k' tR2 [min] k' log k'
4-Nitrobenzamide 1.93 1.52 4.263 1.21 0.0821 4.271 1.21 0.0836
Monuron 1.99 1.46 4.377 1.27 0.103 4.378 1.27 0.103
Aniline 2.07 1.74 5.592 1.90 0.278 5.384 1.79 0.253
2.5-Dichloroaniline 2.55 2.45 5.066 1.62 0.211 5.067 1.62 0.211
Linuron 2.59 2.43 5.427 1.81 0.258 5.437 1.82 0.259
Fenthion 3.31 3.75 6.928 2.59 0.413 6.973 2.61 0.417
Phenanthrene 4.09 4.35 6.656 2.45 0.389 6.706 2.47 0.393
4.4-DDT 5.63 - 9.805 4.08 0.611 9.948 4.15 0.618

tR1 = Retention time 1. Injection; tR2 = Retention time 2. Injection

k’ = Capacity factor; KOC = Adsorption coefficient

The corresponding calibration data for the corresponding calibration lines are summarized in the table below. The calibration data were obtained from the regression line of the graph log k’ vs. log KOC

Calibration Data of the Reference Items for Soil and Sewage Sludge

Soil Sewage Sludge
1st Calibration 2nd Calibration 1st Calibration 2nd Calibration
slope 6.843 6.834 7.742 7.962
intercept 1.0140 1.0230 0.6107 0.5726
r2 0.8619 0.8875 0.7724 0.8261
r 0.9284 0.9421 0.8789 0.9089

r = Correlation Coefficient; r2 = Coefficient



Results of Test Item Analysis – Peak 1

The table below contains the retention time data and calculated log k’ values for Disperse Orange 30. Since two peaks with comparable UV spectra were observed, data were determined for both peaks. Two individual log KOC values for both peaks of the test substance were derived under consideration of the first and second calibration. Finally, a mean value was calculated from these individual log KOC value for soil and for sewage sludge.
Individual log KOC values of 4.35 ± 0.28 (95% CI 3.66 to 5.05) and 4.36 ± 0.26 (95 % CI 3.73 to 4.98) were obtained with both calibrations for the soil and log KOC values of 4.39 ± 0.50 (95 % CI 3.10 to 5.68) and 4.46 ± 0.44 (95 % CI 3.32 to 5.59) for the sewage sludge. The individual values did not differ more than ± 0.25 log KOC values. Based on these data a mean value of 4.36 was derived for soil and 4.43 for sewage sludge. Since the retention time of the test item was higher than that of Phenanthrene for both peaks, the log Koc was extrapolated for sewage sludge

Retention Times, Capacity Factors (k’) and Adsorption Coefficient (log KOC) of Disperse Orange 30 for Soil and Sewage Sludge for Peak 1

log KOC. soil 95% CI log KOC. sewage sludge 95% CI
tR [min] k’ log k’ 1st Calibration 2nd Calibration 1st Calibration 2nd Calibration
7.838 3.06 0.486 4.35 ± 0.28 4.36 ± 0.26 4.39 ± 0.50 4.46 ± 0.44
7.868 3.08 0.488 3.66 to 5.05 3.73 to 4.98 3.10 to 5.68 3.32 to 5.59
7.920 3.10 0.492
Mean 7.88 3.08 0.488 4.36 4.43

tR = Retention time; SD = Standard deviation; CI = Confidence Interval
 
Results of Test Item Analysis – Peak 2

The table below contains the retention time data and calculated log k’ values for Disperse Orange 30. Since two peaks with comparable UV spectra were observed, data were determined for both peaks. Two individual log KOC values for both peaks of the test substance were derived under consideration of the first and second calibration. Finally, a mean value was calculated from these individual log KOC value for soil and for sewage sludge.
Individual log KOC values of 5.13 ± 0.39 (95% CI 4.17 to 6.08) and 5.13 ± 0.35 (95% CI 4.28 to 5.98) were obtained with both calibrations for the soil and log KOC values of 5.26 ± 0.70 (95% CI 3.46 to 7.06) and 5.36 ± 0.61 (95% CI 3.78 to 6.93) for the sewage sludge. The individual values did not differ more than ± 0.25 log KOC values. Based on these data a mean value of 5.13 was derived for soil and 5.31 for sewage sludge. Since the mean retention time for the test item was higher than for Phenantrene, the log KOC values were extrapolated for sewage sludge.

Retention Times, Capacity Factors (k’) and Adsorption Coefficient (log KOC) of Disperse Orange 30 for Soil and Sewage Sludge for Peak 2

log KOC. soil 95% CI log KOC. sewage sludge 95% CI
tR [min] k’ log k’ 1st Calibration 2nd Calibration 1st Calibration 2nd Calibration
9.566 3.95 0.597 5.13 ± 0.39 5.13 ± 0.35 5.26 ± 0.70 5.36 ± 0.61
9.636 3.99 0.601 4.17 to 6.08 4.28 to 5.98 3.46 to 7.06 3.78 to 6.93
9.709 4.03 0.605
Mean 9.64 3.99 0.601 5.13 5.31

Validity criteria fulfilled:

yes

Remarks:

The value of log KOC felt within a range of ± 0.25 log units. The variation in temperature was between ± 1.1°C. A signal-to-noise ratio > 3 was provided for NaNO3, reference and the test items.

Conclusions:

Under the study conditions, the adsorption coefficients determined for the different peaks were as follows: for peak 1, log KOC of 4.36 for soil and log KOC of 4.43 for sewage sludge and for peak 2, log KOC of 5.13 for soil and log KOC of 5.31 for sewage sludge.

Executive summary:

The adsorption coefficients of the test substance in soil and in sewage sludge were determined using high performance liquid chromatography (HPLC) according to OECD Guideline 121 and EU Method C.19, in compliance with GLP.The test was performed at one pH value since the test substance is not ionisable in the relevant pH range 5.5 to 7.5 and temperature (21.8°C, mean value). A 10 mg/L sample was analyzed three-fold via HPLC on a cyanopropyl (CN-) column with DAD detection.Calibrations were performed using eight reference substances for soil and seven substances for sewage sludge with known log Koc values (given by the guidelines). The reference substances were measured twice, once before and once after the measurement (double injection of one replicate) of the test substance. Two individual log Koc values for soil and for sewage sludge were derived under consideration of the first and second calibration. Two peaks with comparable UV spectra were determined and the log Koc values were calculated for both. Since the retention time of the test substance was higher than that of the reference substance phenanthrene for both peaks, the log Koc was extrapolated for sewage sludge. Under the study conditions, the adsorption coefficients determined for the different peaks were as follows: for peak 1, log Koc of 4.36 for soil and log Koc of 4.43 for sewage sludge and for peak 2, log Koc of 5.13 for soil and log Koc of 5.31 for sewage sludge (Landsberg, 2016).

Key value for chemical safety assessment

Koc at 20 °C:

22 908.6

Additional information

The adsorption coefficients of the test substance in soil and in sewage sludge were determined using high performance liquid chromatography (HPLC) according to OECD Guideline 121 and EU Method C.19, in compliance with GLP.The test was performed at one pH value since the test substance is not ionisable in the relevant pH range 5.5 to 7.5 and temperature (21.8°C, mean value). A 10 mg/L sample was analyzed three-fold via HPLC on a cyanopropyl (CN-) column with DAD detection. Calibrations were performed using eight reference substances for soil and seven substances for sewage sludge with known log Koc values (given by the guidelines). The reference substances were measured twice, once before and once after the measurement (double injection of one replicate) of the test substance. Two individual log Koc values for soil and for sewage sludge were derived under consideration of the first and second calibration. Two peaks with comparable UV spectra were determined and the log Koc values were calculated for both. Since the retention time of the test substance was higher than that of the reference substance phenanthrene for both peaks, the log Koc was extrapolated for sewage sludge. Under the study conditions, the adsorption coefficients determined for the different peaks were as follows: for peak 1, log Koc of 4.36 for soil and log Koc of 4.43 for sewage sludge and for peak 2, log Koc of 5.13 for soil and log Koc of 5.31 for sewage sludge (Landsberg, 2016).

[LogKoc: 4.36]