4-[[2-methoxy-4-[(4-nitrophenyl)azo]phenyl]azo]phenol

Adsorption / desorption

Administrative data

Endpoint:

adsorption / desorption: screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-10-18 to 2016-10-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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))

Deviations:

no

GLP compliance:

yes

Type of method:

HPLC estimation method

Media:

soil/sewage sludge

Test material

Test material information

Constituent 1

Reference substance name:

4-[[2-methoxy-4-[(4-nitrophenyl)azo]phenyl]azo]phenol

EC Number:

243-325-5

EC Name:

4-[[2-methoxy-4-[(4-nitrophenyl)azo]phenyl]azo]phenol

Cas Number:

19800-42-1

Molecular formula:

C19H15N5O4

IUPAC Name:

4-[[2-methoxy-4-[(4-nitrophenyl)azo]phenyl]azo]phenol

Test material form:

solid: particulate/powder

Remarks:

Dark brown

Radiolabelling:

no

Study design

Test temperature:

The column temperature during the study was monitored every 2 minutes by a data logger. A mean value of 21.1 °C at pH 5.5 and 20.8 at pH 7.5 was obtained for the temperature during the course of the study.

HPLC method

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 : KH2PO4 buffer (pH 7.5) (70 :30 v/v) and Methanol : KH2PO4 buffer (pH 5.5) (70 :30 v/v) were 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 will be 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 1. Methanol : KH2PO4 buffer (pH 5.5) (70 : 30 v/v)

2. Methanol : KH2PO4 buffer (pH 7.5) (70 : 30 v/v)

The study was performed under two different pH conditions. Based on calculation with chemicalize.org the test item is ionisised > 10% in the relevant pH range from 5.5 to 7.5.

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
pH meter, Multi 350i, WTW
Laboratory glassware

Reagents HPLC water, for HPLC, batch 16H184002, VWR
Methanol, gradient grade, batch 16G224004, VWR
Potassium dihydrogenphosphate (KH2PO4), 99.9%, batch 15H200005, VWR
Sodium phosphate dibasic dihydrate (Na2HPO4 * 2H2O), ≥ 98 %, batch BCP4525V, FLUKA
Sodium hydroxide, > 97%, batch: 16B290018, VWR
Phosphoric acid, ≥ 85 %, batch 235229239, ROTH
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 minutes interval

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

Software The data presented in the tables were computer-generated and rounded for presentation. Thus manual calculation of results based on the data in this report may yield minor deviations from these figures.
Calculations were carried out using the software:
• Microsoft® Office Excel, MICROSOFT CORPORATION
• GraphPadPrism, GRAPHPAD SOFTWARE, INC

Batch equilibrium or other method

Analytical monitoring:

not required

Details on sampling:

TEST ITEM Disperse Orange 29

Stock solution 100 mg/L in methanol

Test concentration 10 mg/L
Dilution With methanol and phosphate buffer 5.5 and methanol and phosphate buffer 7.5, respectively

Replicates Single (three injections)

Detection With DAD

Wavelength Detected range: 210nm – 500nm
Data evaluated at 418 nm at pH 5.5 and 415 nm at pH 7.5

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 phosphate buffer 5.5 and methanol and phosphate buffer 7.5, respectively

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

Results and discussion

Adsorption coefficient

Adsorption coefficient 1

Key result

Type:

log Koc

Value:

ca. 5.65 dimensionless

pH:

5.5

Temp.:

21.1 °C

Matrix:

soil

Adsorption coefficient 2

Key result

Type:

log Koc

Value:

ca. 5.93 dimensionless

pH:

5.5

Temp.:

21.1 °C

Matrix:

sewage sludge

Adsorption coefficient 3

Key result

Type:

log Koc

Value:

ca. 3.52 dimensionless

pH:

7.5

Temp.:

20.8 °C

Matrix:

soil

Adsorption coefficient 4

Key result

Type:

log Koc

Value:

ca. 3.51 dimensionless

pH:

7.5

Temp.:

20.8 °C

Matrix:

sewage sludge

Results: HPLC method

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
Determination at pH 5.5 Determination at pH 7.5
Min. Temperature 20.1 °C 20.2 °C
Max. Temperature 23.1 °C 22.0 °C
Mean Value 21.1 °C 20.8 °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 Determination at pH 7.5
t0 t0
[min] [min]
1 2.218 1.969
2 2.236 1.966
3 2.243 1.969
Mean ± SD 2.23 +/- 0.01 1.968 +/- 0.002
SD = Standard deviation

Calibration Data

Determination at pH 5.5
Retention time data of the reference items used for calibration of soil and sewage sludge at pH 5.5 are given in the table below.

Capacity Factors (k’) of the Reference Items for Soil and Sewage Sludge at pH 5.5
Reference Item log KOC. soil log KOC. sewage sludge 1st Calibration 2nd Calibration
tR1 [min] k' log k' tR2 [min] k' log k'
4-Nitrobenzamide 1.93 1.52 3.879 0.738 -0.132 0.740 -0.131 1.93
Monuron 1.99 1.46 4.053 0.816 -0.0885 0.818 -0.0871 1.99
Aniline 2.07 1.74 3.747 0.679 -0.168 0.685 -0.164 2.07
Linuron 2.59 2.43 4.863 1.18 0.0713 1.18 0.0711 2.59
Phenylbenzoate 2.87 3.26 4.793 1.15 0.0596 1.14 0.0564 2.87
Fenthion 3.31 3.75 6.070 1.72 0.235 1.70 0.230 3.31
Phenanthrene 4.09 4.35 6.066 1.72 0.235 1.69 0.228 4.09
4.4-DDT 5.63 - 8.323 2.73 0.436 2.70 0.431 5.63
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 at pH 5.5 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 at pH 5.5
Soil Sewage Sludge
1st Calibration 2nd Calibration 1st Calibration 2nd Calibration
slope 5.761 5.861 6.545 6.682
intercept 2.593 2.595 2.446 2.45
r2 0.904 0.9055 0.8849 0.882
r 0.9508 0.9515 0.9407 0.9391
R = Correlation Coefficient; r2 = Coefficient of Determination



Determination at pH 7.5
Retention time data of the reference items used for calibration of soil and sewage sludge at pH 7.5 are given in the table below.

Capacity Factors (k’) of the Reference Items for Soil and Sewage Sludge at pH 7.5
Reference Item log KOC. soil log KOC. sewage sludge 1st Calibration 2nd Calibration
tR1 [min] k' log k' tR2 [min] k' log k'
4-Nitrobenzamide 1.93 1.52 3.860 0.961 -0.0171 3.866 0.964 -0.0157
Monuron 1.99 1.46 4.037 1.05 0.0217 4.041 1.05 0.0226
Aniline 2.07 1.74 3.741 0.901 -0.0453 3.738 0.899 -0.0461
Linuron 2.59 2.43 4.830 1.45 0.163 4.816 1.45 0.161
Phenylbenzoate 2.87 3.26 4.797 1.44 0.158 4.786 1.43 0.156
Fenthion 3.31 3.75 6.022 2.06 0.314 6.016 2.06 0.313
Phenanthrene 4.09 4.35 6.019 2.06 0.314 6.064 2.08 0.318
4.4-DDT 5.63 - 8.219 3.18 0.502 8.235 3.18 0.503
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 at pH 7.5 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 at pH 7.5
Soil ewage Sludge
1st Calibration 2nd Calibration 1st Calibration 2nd Calibration
slope 6.380 6.374 7.328 7.320
intercept 1.935 1.935 1.693 1.694
r2 0.9112 0.9138 0.8927 0.8944
r 0.9546 0.9559 0.9448 0.9457
R = Correlation Coefficient; r2 = Coefficient of Determination



Results of Test Item Analysis at pH 5.5

The table below contains the retention time data and calculated log k’ values for Disperse Orange 29 at pH 5.5. Two individual log KOC values for the test item 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 5.62 ± 0.37 (95 % CI 4.71 to 6.54) and 5.68 ± 0.38 (95 % CI 4.76 to 6.60) were obtained with both calibrations for the soil and log KOC values of 5.89 ± 0.55 (95 % CI 4.48 to 7.30) and 5.97 ± 0.57 (95 % CI 4.51 to 7.42) 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 5.65 was derived for soil and 5.93 for sewage sludge. Since the mean retention time for the test item was 9.73 min at pH 5.5, these values are extrapolated because the retention time of the test item was higher than that of the reference item with the highest log KOC.

Retention Times, Capacity Factors (k’) and Adsorption Coefficient (log KOC) of Disperse Orange 29 for Soil and Sewage Sludge at pH 5.5

log KOC. soil log KOC. sewage sludge
tR [min] k’ log k’ 95 % CI 95 % CI
1st Calibration 2nd Calibration 1st Calibration 2nd Calibration
9.698 3.34 0.524 5.62 ± 0.37 5.68 ± 0.38 5.89 ± 0.55 5.97 ± 0.57
9.744 3.36 0.527 4.71 to 6.54 4.76 to 6.60 4.48 to 7.30 4.51 to 7.42
9.746 3.37 0.527
Mean 9.73 3.36 0.526 5.65 5.93
tR = Retention time; SD = Standard deviation; CI = Confidence Interval
 

Results of Test Item Analysis at pH 7.5

The table below contains the retention time data and calculated log k’ values for Disperse Orange 29 at pH 7.5. Two individual log KOC values for the test item 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 3.52 ± 0.16 (95 % CI 3.14 to 3.90) and 3.52 ± 0.15 (95 % CI 3.14 to 3.89) were obtained with both calibrations for the soil and log KOC values of 3.51 ± 0.21 (95 % CI 2.98 to 4.04) 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 3.52 was derived for soil and 3.51 for sewage sludge.

Retention Times, Capacity Factors (k’) and Adsorption Coefficient (log KOC) of Disperse Orange 29 for Soil and Sewage Sludge at pH 7.5

log KOC. soil log KOC. sewage sludge
tR [min] k’ log k’ 95 % CI 95 % CI
1st Calibration 2nd Calibration 1st Calibration 2nd Calibration
5.452 1.77 0.248 3.52 ± 0.16 3.52 ± 0.15 3.51 ± 0.21 3.51 ± 0.21
5.435 1.76 0.246 3.14 to 3.90 3.14 to 3.89 2.98 to 4.04 2.98 to 4.04
5.470 1.78 0.250
Mean 5.45 1.77 0.248 3.52 3.51
tR = Retention time; SD = Standard deviation; CI = Confidence Interval

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Calculated values for log KOC of the test item did not differ by more than 0.25 between injections. The variation in temperature was between ± 1 °C during the course of measurements. A signal-to-noise ratio > 3 was provided for all substances.

Conclusions:

The adsorption coefficient log Koc of the test substance was determined to be 5.65 for soil and 5.93 for sewage sludge at pH 5.5 (21.1 °C) and 3.52 for soil and 3.51 for sewage sludge at pH 7.5 (20.8 °C).

Executive summary:

A study was conducted to determine the adsorption coefficient (Koc) of the test substance (94.8% purity) on soil and on sewage sludge using high performance liquid chromatography (HPLC) according to OECD Guideline 121 and EU Method C.19. Two pH values were used since the test substance is ionisable in the relevant pH range of 5.5 to 7.5. The temperature was controlled (21.1 °C for the buffer 5.5 and 20.8 °C for the buffer 7.5, mean value). A methanol : KH2PO4(pH 5.5) (70 : 30 v/v) and a methanol : KH2PO4 buffer (pH 7.5) (70 : 30 v/v) were used as mobile phases. A 10mg/L sample of the test substance was analysed threefold via HPLC on a cyanopropyl (CN-) column with DAD detection. Calibrations were performed using eight reference items for soil and seven reference items for sewage sludge with known log Koc 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. Dead time of the HPLC-system was determined with sodium nitrate. Two individual log Koc values for soil and for sewage sludge were derived under consideration of the first and second calibration at each pH value. Finally, a mean value for soil and for sewage sludge was calculated from these individual log Koc values. The log Koc values were determined to be 5.65 for soil and 5.93 for sewage sludge at pH 5.5 (21.1 °C). Values at pH 7.5, extrapolated because the retention time of the test item was higher than that of the reference item with the highest log Koc, were equivalent to 3.52 for soil and 3.51 for sewage sludge (20.8 °C) (Landsberg, 2016).