[4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate

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:

From 23-03-2023 to 30-03-2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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))

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))

GLP compliance:

yes

Type of method:

HPLC estimation method

Test temperature:

20 to 25 ± 1 °C

Details on study design: HPLC method:

EQUIPMENT
- Apparatus: Analytical HPLC-column packed with a cyanopropyl stationary phase and coupled to a diode array detector (DAD).
- Type: HPLC AGILENT 1100 consisting of:
Auto sampler G1329A
Auto sampler thermostat G1330B
Pump G1312A
Degasser G1322A
Column oven G1316A
Detector G1315B

Software: OpenLab 2.2, AGILENT TECHNOLOGIES

Injection volume: 20 µL

Flow rate: 1.0 mL/min

Column temperature : 20 to 25 ± 1 °C

- Type, material and dimension of analytical (guard) column: HPLC-column: YMC Pack CN / S 5 µm, 250 x 4 mm, YMC EUROPE GMBH
- Detection system: Diode array detection (DAD)

MOBILE PHASES
- Type: Methanol : buffer (70 : 30 v/v) for pH 5.5 and (85 : 15 v/v) for pH 7.5 will be used as mobile phase. The test will be performed at buffered pH conditions of 5.5 and 7.5, respectively.
- Experiments with additives carried out on separate columns: no
- pH: The test was performed at buffered pH conditions of 5.5 and 7.5, respectively.

REFERENCE SUBSTANCES
- Identity: Sodium nitrate will be used for determination of void time. 3-Nitrobenzamide and Endosulfan-diol will be used for approval of the system suitability.
As reference items, the following chemicals acc. to the guidelines will be used:
Sodium nitrate 7631-99-4,
3-Nitrobenzamide 645-09-0
Endosulfan-diol 2157-19-9
Acetanilide 103-84-4
Atrazine 1912-24-9
Monuron 150-68-5
Linuron 330-55-2
Fenthion 55-38-9
Phenanthrene 85-01-8
4,4’-DDT 50-29-3


DETERMINATION OF RETENTION TIMES
- Quantity of test substance introduced in the column: 10 mg/L
- Quantity of reference substances: 20 mg/L for Endosulfan diol, 10 mg/L the others
- Intervals of calibration: Determination of retention times tR of at least six reference items (one calibration before and one after test item injection

REPETITIONS
- Number of determinations: Determination of the retention time of the test item with two injections

EVALUATION
- Calculation of capacity factors k':

Calibration of the method will be done by determination retention times tR of the reference items (minimum of six items). Each reference item will be analysed separately once before and after the test item.

The retention time will be described by the capacity factor k’ given by the expression:

k'=(tR-t0)/t0 (1)

k’: capacity factor, tR: retention time, t0: void time


- Calculation of retention times:

Determination of the retention time of the test item with two injections

- Determination of the log Koc value:

By linear regression of the log KOC of the reference items against the log k’ of the reference items the equation for two calibration graphs will be obtained.

Two individual adsorption coefficients for the test item will be calculated by experimentally determining its capacity factor k´ and inserting k´ into the following equation (2) obtained from the first and second calibration graph:

log⁡KOC=a∙log⁡k+b (2)

a: slope of the calibration graph
b: y-intercept of calibration graph

Finally, a mean value will be calculated from these individual log KOC values.

Key result

Sample No.:

#1

Type:

log Koc

Remarks:

Log KOC soil

Value:

10.6 - 10.61

pH:

5.5

Temp.:

22.7 °C

Key result

Sample No.:

#2

Type:

log Koc

Remarks:

LOG KOC sewage sludge

Value:

10.62 - 10.64

pH:

5.5

Temp.:

22.7 °C

Key result

Sample No.:

#3

Type:

log Koc

Remarks:

log KOC soil

Value:

47.44 - 47.9

pH:

7.5

Temp.:

22.5 °C

Key result

Sample No.:

#4

Type:

log Koc

Remarks:

log Koc sewage sludge

Value:

55.16 - 55.9

pH:

7.5

Temp.:

22.5 °C

Details on results (HPLC method):

- Retention times of reference substances used for calibration: see below
- Details of fitted regression line (log k' vs. log Koc): see below
- Graph of regression line attached : see attached document
- Average retention data for test substance: see below

Retention times of reference substances used for calibration:

• pH 5.5

Table 7: Calibration Data of the Reference Items for Soil and Sewage Sludge at pH 5.5

Reference Item	log KOC soil	log KOC sewage sludge	1^st Calibration			2^nd Calibration
			tR1 [min]	k'	log k'	tR2 [min]	k'	log k'
Acetanilide	1.26	1.52	2.859	0.415	-0.382	2.854	0.412	-0.385
Atrazine	1.81	1.66	2.943	0.456	-0.341	2.937	0.453	-0.343
Monuron	1.99	1.46	3.027	0.498	-0.303	3.022	0.496	-0.305
Linuron	2.59	2.43	3.314	0.640	-0.194	3.309	0.638	-0.195
Fenthion	3.31	3.75	3.716	0.839	-0.0762	3.710	0.836	-0.0778
Phenanthrene	4.09	4.35	3.829	0.895	-0.0482	3.823	0.892	-0.0497
4,4‘-DDT	5.63	-	4.524	1.24	0.0930	4.523	1.24	0.0929

t_R1 = Retention time 1. Injection,

t_R2 = Retention time 2. Injection,

k’       = Capacity factor,

K_OC     = Adsorption coefficient

 

• pH : 7.5

Table 12: Calibration Data of the Reference Items for Soil and Sewage Sludge at pH 7.5

Reference Item	log KOC soil	log KOC sewage sludge	1^st Calibration			2^nd Calibration
			tR1 [min]	k'	log k'	tR2 [min]	k'	log k'
Acetanilide	1.26	1.52	2.682	0.735	-0.134	2.669	0.726	-0.139
Atrazine	1.81	1.66	2.666	0.724	-0.140	2.643	0.710	-0.149
Monuron	1.99	1.46	2.732	0.767	-0.115	2.718	0.758	-0.120
Linuron	2.59	2.43	2.787	0.803	-0.0954	2.774	0.794	-0.100
Fenthion	3.31	3.75	2.877	0.861	-0.0650	2.852	0.845	-0.0733
Phenanthrene	4.09	4.35	2.937	0.900	-0.0459	2.920	0.889	-0.0512
4,4‘-DDT^1	5.63	-	2.926	0.893	-0.0493	2.903	0.878	-0.0566

t_R1     = Retention time 1. Injection

t_R2     = Retention time 2. Injection

k’       = Capacity factor

K_OC   = Adsorption coefficient

1)      = Determined to be an outlier via F-test, not taken into account

 

Details of fitted regression line (log k' vs. log Koc)

• pH = 5.5

The calculated slope, Y-intercept and correlation coefficient for the first and second calibration of the reference items for soil and sewage sludge are shown in Table 8. The correlation coefficient r for all calibrations was > 0.9

Table 8: Slope, Y-intercept and Correlation Coefficient of the Regression Lines for Soil and Sewage Sludge at pH 5.5

	Soil		Sewage Sludge
	1^st Calibration	2^nd Calibration	1^st Calibration	2^nd Calibration
slope	8.485	8.449	8.556	8.522
intercept	4.471	4.478	4.446	4.453
r^2	0.9623	0.9626	0.9330	0.9317
r	0.9810	0.9811	0.9659	0.9652

r = Correlation Coefficient
r² = Coefficient

• pH = 7.5

The calculated slope, Y-intercept and correlation coefficient for the first and second calibration of the reference items for soil and sewage sludge are shown in Table 13. The correlation coefficient r for all calibrations was > 0.9.

	Soil		Sewage Sludge
	1^st Calibration	2^nd Calibration	1^st Calibration	2^nd Calibration
slope	26.87	26.50	31.59	31.04
intercept	5.175	5.302	5.663	5.801
r^2	0.9448	0.9302	0.9229	0.9023
r	0.9720	0.9645	0.9607	0.9499

r  = Correlation Coefficient
r²   = Coefficient

Average retention data for test substance

• pH = 5.5

Table 9:  Retention Times, Capacity Factors and Adsorption Coefficient of Test Item at pH 5.5

Test Item	tR [min]	k’	log k’	log KOC. soil ± SD 95% CI		log KOC. sewage sludge ± SD 95% CI
				1^st Calibration	2^nd Calibration	1^st Calibration	2^nd Calibration
	12.637	5.25	0.720	10.61 ± 0.69 8.84 to 12.38	10.60 ± 0.68 8.84 to 12.35	10.64 ± 1.10 7.60 to 13.69	10.62 ± 1.11 7.55 to 13.69
	12.746	5.31	0.725
	12.804	5.34	0.727
Mean	12.729	5.30	0.724	10.61		10.63

t_R             = Retention time (corrected with the difference between the respective detectors)
K_OC          = Adsorption coefficient according to the guidelines

CI            = Confidence Interval

• pH= 7.5

Table 14: Retention Times, Capacity Factors and Adsorption Coefficient of Test Item at pH 7.5

Test Item	tR [min]	k’	log k’	log KOC. soil ± SD 95% CI		log KOC. sewage sludge ± SD 95% CI
				1^st Calibration	2^nd Calibration	1^st Calibration	2^nd Calibration
	56.047	35.3	1.55	47.90 ± 5.49 32.67 to 63.144	47.44 ± 6.16 30.35 to 64.52	55.90 ± 7.71 34.49 to 77.30	55.16 ± 8.66 31.11 to 79.21
	63.775	40.3	1.60
	64.686	40.8	1.61
Mean	61.503	38.8	1.59	47.67		55.53

 

Validity criteria fulfilled:

yes

Remarks:

Sensitivity : fulfilled Repeatability fulfilled for pH = 5.5, not fulfilled for pH 7.5 (this was deemed acceptable as the corresponding values were highly extrapolated) System suitability : fulfilled Temperature : fulfilled

Conclusions:

The adsorption coefficients (log KOC) of the test item were determined for pH 5.5 at a mean temperature of 22.7 °C to be 10.61 for soil as well as 10.63 for sewage sludge and for pH 7.5 at a mean temperature of 22.5 °C to be 47.67 for soil as well as 55.53 for sewage sludge, respectively. Because the test item eluted after the reference items with the highest log KOC value, the log KOC values of the test item were extrapolated from the calibration data.

Executive summary:

The adsorption coefficient (K_OC) for the test item  (batch number: 924249) on soil and on sewage sludge using high performance liquid chromatography (HPLC) was estimated according to OECD Guideline 121 (2001) and Council Regulation (EC) No. 440/2008, Method C.19.

For each pH condition, pH 5.5 and 7.5, calibrations were performed using seven reference items for soil and six reference items for 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 (three injections of one replicate) and were detected by DAD. Sodium nitrate was used for the determination of the void time of the HPLC system coupled to DAD. For calibration data and detailed results please refer to section 7. Because the test item eluted after the reference item with the highest log K_OC value, the values of the test item were extrapolated from the calibration data.

Two individual log K_OC values for soil and for sewage sludge for each of the observed test item were derived under consideration of the first and second calibration.

The adsorption coefficients (log K_OC) of the test item signals were determined for pH 5.5 (22.7 °C) to be for soil : 10.61 (mean value) and for sewage sludge: 10.63 (mean value)

The adsorption coefficients (log K_OC) of the test item signals were determined for pH 7.5 (22.5 °C) to be for soil : 47.67 (mean value) and for sewage sludge: 55.53 (mean value)

Description of key information

Selection of the Koc value to perform the CSA, determined according to OECD 121

Key value for chemical safety assessment

Koc at 20 °C:

40 700 000 000

Additional information

The adsorption coefficient (K_OC) of the test item on soil and on sewage sludge using high performance liquid chromatography (HPLC) was estimated according to OECD Guideline 121 at two pHs : 5.5 and 7.5.

For each pH condition, pH 5.5 and 7.5, calibrations were performed using seven reference items for soil and six reference items for 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 (three injections of one replicate) and were detected by DAD. Two individual log K_OC values for soil and for sewage sludge for each of the observed test item were derived under consideration of the first and second calibration. Because the test item eluted after the reference item with the highest log K_OC value, the values of the test item were extrapolated from the calibration data.

The adsorption coefficients (log K_OC) of the test item signals were determined for pH 5.5 (22.7 °C) to be for soil: 10.61 and for sewage sludge:10.63

In the chemical safety report, the mean KOC value obtained for soil at pH 5.5, 4,07E10 , representing a worst case scenario, is used to determine the PEC.

 

Given the high calculated log Koc value, it indicates that the substance would adsorb rapidly to soil or sludge, showing little or no mobility (according to P.J. McCall et al., 1981)¹ and therefore low to no presence in the aquatic compartment of the environment.

¹ McCall P.J., Laskowski D.A., Swann R.L., and Dishburger H.J., (1981), “Measurement of sorption coefficients of organic chemicals and their use, in environmental fate analysis”, in Test Protocols for Environmental Fate and Movement of Toxicants. Proceedings of AOAC Symposium, AOAC, washington DC.

McCall classification scheme of mobility

 

Koc range	Class of mobility
0 - 50	Very high
50 - 150	High
150 - 500	Medium
500 - 2000	Low
2000 - 5000	Slightly
> 5000	Immobile