Reaction mass of 6-amino-2-anilino-4-methyl-5-{(E)-[4-nitro-2-(substituted)phenyl]diazenyl}nicotinonitrile and 2-amino-6-anilino-4-methyl-5-{(E)-[4-nitro-2-(substituted)phenyl]diazenyl}heteromonocyclic nitrile

Adsorption / desorption

Link to relevant study record(s)

Reference

Endpoint:

adsorption / desorption: screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 January 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and EC guidelines and according to the Principles of Good Laboratory Practice (GLP).

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))

Version / remarks:

(2008)

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:

(2001)

Principles of method if other than guideline:

The HPLC method using soil-adsorption-reference data was applied for the determination of the adsorption coefficient (Koc) of FAT 41043/A.

Principle of the HPLC method: While passing through the column along with the mobile phase the test substance interacts with the stationary phase. As a result of partitioning between mobile and stationary phases, the test substance is retarded. The dual composition of a cyanopropyl stationary phase, having polar and non-polar sites allows for interaction of polar and non-polar groups of a molecule in a similar way as is the case for organic matter in soil or sewage sludge matrices. This enables the relationship between the retention time on the column and the Kocon organic matter to be established.

GLP compliance:

yes

Type of method:

HPLC estimation method

Media:

other: mobile phase at pH 7

Radiolabelling:

no

Details on study design: HPLC method:

CALCULATION of PKA
According to the guidelines, the determination of the Koc for test substances that are ionized for at least 10% within pH 5.5 to 7.5 should be performed with both the ionized and non-ionized form. Therefore, the pKa values of the test substance were calculated using the Perrin calculation method (pKalc 5.0, module in Pallas 3.0, CompuDrug International San Francisco, CA, USA). The following pKa values in the logarithm range of 1 - 14 for acidic and basic groups in the molecular structure of the test substance were calculated using the Perrin calculation method:
pKa Acidic: None
pKa Basic: 3.8 (Pyridinium)
Based on the calculations, the HPLC method was performed at pH 7.


ANALYTICAL CONDITIONS HPLC METHOD:
Instrument: Alliance Separation Module 2695 (Waters, Milford, MA, USA)
Detector: Dual λ Absorbance Detector 2487 (Waters)
Column: Hypersil-CN, 150 mmx4.6 mm i.d., dp = 5 µm (Thermo Fisher Scientific, Waltham, MA, USA)
Column temperature: 35 °C ± 1 °C
Mobile phase: 55/45 (v/v) methanol/0.1 M phosphate buffer pH 7*
Flow: 1 ml/min
Injection volume: 10 µl
UV detection: 210 nm

*Phosphate buffer pH 7, 0.1 M :   0.1 M potassium dihydrogenphosphate solution adjusted to pH 7 using 10 N sodium hydroxide.The buffer contains 0.0009 % (w/v) sodium azide.


REFERENCE SUBSTANCES:
Solutions of reference substances with known log Koc values based on soil adsorption and the test substance were analysed.

Reference substance Purity CAS number log Koc#

Acetanilide > 99.9 % 103-84-4 1.26
Atrazine 97.5 % 1912-24-9 1.81
Monuron 99.9 % 150-68-5 1.99
2,5-Dichloroaniline 99.8 % 95-82-9 2.55
Benzoic acid phenylester 99.9 % 93-99-2 2.87
Fenthion 98.3 % 55-38-9 3.31
Phenanthrene 98.1 % 85-01-8 4.09
4,4’-DDT 98.2 % 50-29-3 5.63

# values according to the OECD 121 guideline based on soil adsorption data

 
PREPARATION OF SOLUTIONS
Solution of the unretained compound: 4.81 g/l stock solution of formamide (99.8 %, [CAS#75-12-7]) in methanol was used. The stock solution was diluted to obtain an end solution of 55/45 (v/v) methanol/phosphate buffer pH 7. The formamide blank solution was 55/45 (v/v) methanol/phosphate buffer pH 7.
 
Reference substance solutions: Stock solutions of the reference substances at concentrations of approximately 1 g/l in methanol were used. The stock solutions were diluted to obtain an end solution of 55/45 (v/v) methanol/phosphate buffer pH 7. The blank solution for the mixture of reference substances was 55/45 (v/v) methanol/phosphate buffer pH 7.

Test solution: A 1815 mg/l stock solution of the test substance was prepared in acetone. In order to dissolve the test substance the stock solution was ultrasonicated for 5 minutes. The stock solution was 100-times diluted with 75/25 (v/v) methanol/phosphate buffer pH 7[1].The final concentration of the test substance solutions was 18.2 mg/l.
[1]   It was observed that the test substance precipitates when 100-times diluted in 55/45 (v/v) methanol/phosphate buffer pH 7. With an additional run at an injection volume of 2 µl it was proved that the end matrix of the test solution had no effect on the retention time of the test substance.


PERFORMANCE OF THE TEST:
The reference substance and test substance solutions were injected in duplicate. Blank solutions were analysed by single injection. The capacity factor (k') of each compound was calculated from its retention time. The log k’ values of the references substances were plotted against the known log Koc values. A linear regression program was used to calculate the calibration curve. Linear regression analysis was performed using the least squares method. The coefficient of correlation (r) was calculated. The log Koc value for the test substance was calculated by substituting its mean log k’ in the calibration curve. The value of log Koc obtained from duplicate measurements was within ±0.25 log units.

Type:

log Koc

Value:

6.42

Temp.:

35 °C

Remarks on result:

other: The two isomers of the test substance were detected as a single peak

Details on results (HPLC method):

In the chromatogram of the test solution, one test substance peak was observed. It was considered that the two isomers of the test substance eluted as a single peak at 10.2 minutes. HPLC chromatograms of the test substance solution and corresponding blank are shown in Figure 1. With a gradient to 100% methanol performed in a preliminary test it was demonstrated that no compounds related to the test substance eluted after 15 minutes (results are archived in the raw data).

The results of the HPLC method are given in Table 1. Figure 2 the shows the calibration curve of the log k’ of the reference substances as function of log Koc. The equation of the regression line was: log k’ = 0.273´log Koc– 1.13 (r = 0.98, n = 16).

Table1            K_ocof the test substance

Substance	tr,1 [min]	tr,2 [min]	mean tr (n=2)	log Koc	Koc	Area %
Formamide (t0)	1.963	1.964	1.964
Acetanilide	2.220	2.216		1.26
Atrazine	2.422	2.415		1.81
Monuron	2.446	2.436		1.99
2,5-Dichloroaniline	2.669	2.652		2.55
Benzoic acid phenylester	3.023	3.001		2.87
Fenthion	3.643	3.598		3.31
Phenanthrene	3.799	3.755		4.09
4,4’-DDT	6.169	6.049		5.63
Test substance	10.200	10.167	10.184	6.42	2.6x10^6	100

 

 

 

 

 

Conclusions:

The Koc of FAT 41043/A was determined to be 2.6 x 10E6 (log Koc= 6.42) by the HPLC estimation method.

Description of key information

The Koc of FAT 41043/A was determined to be 2.6 x 10E6 (log Koc= 6.42) by the HPLC estimation method (EC C.19 and OECD 121).

Additional information