Reaction Mass of Cyclohexanepropanol, 2,2,6-trimethyla-propyl-, (alpha.R,1R,6S)- and Cyclohexanepropanol,2,2,6-trimethyl-a-propyl-, [1a(S*),6b]- (9CI)

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 4 April 2019 to 30 April 2019

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Dates of inspection: 29-30-31 May 2018 / Date of signature: 15 November 2018

Type of method:

HPLC estimation method

Media:

soil/sewage sludge

Specific details on test material used for the study:

- pKa: Based on chemical structure (secondary alcohol), no ionisation anticipated in environmental relevant pH range

Radiolabelling:

no

Test temperature:

The temperature variations of the oven containing the HPLC column were included in the range 29.4 °C – 30.7 °C.

Details on study design: HPLC method:

EQUIPMENT
- HPLC-MSMS: WATERS 2695, Quattro micro API
- Column: Roc Cyano (4.6 x 150 mm, 5 μm), RESTEK; injection volume 100 µL; column flow 1 mL/min; column temperature 30 +/- 1°C; duration 25 minutes
- LC method: log Koc
- MS method:l og Koc fullscan pos; log Koc fullscan neg
- MS parameters: Cone voltage 20 V; Mass 50 - 500; Scan time 0.75; Inter-scan delay 0.3; Ionisation mode Electrospray negative ES- (log Koc fullscan neg) or Electrospray positive ES+ (log Koc fullscan pos)

MOBILE PHASES
- Type: Methanol: 55 %; Ultrapure water: 45 %
The pH value measured in the ultrapure water used for the mobile phase was to 6.71.

PREPARATION OF THE TEST ITEM SOLUTION
A stock solution of the test item was prepared at a concentration of 10.26 g/L in acetone. A diluted solution at 102.6 mg/L was prepared in the HPLC mobile phase. Sodium nitrate at 1 mg/L was added to this solution. These concentrations were adjusted depending on the response intensity of the compound on the system. The diluted solution was injected in the analytical system with the electrospray negative (ES-) mode

PREPARATION OF THE REFERENCE SUBSTANCES SOLUTIONS
Concerning the reference substances solutions prepared from pure analytical standard, a stock solution of each reference substance was prepared at a concentration of approximately 1 g/L in acetone. These solutions were kept at 1 - 5 °C for 6 months. Two diluted reference solutions (MIX POS and MIX NEG) containing all the reference substances were prepared in the HPLC mobile phase. The MIX POS was injected with the electrospray positive mode and the MIX NEG was injected with the electrospray negative mode. The concentrations of the reference substances were adjusted depending on the response intensity of each compound on the system according to the table 5.4.1/1 in "Any other information on materials and methods incl. tables". Sodium nitrate at 1 mg/L was added to the MIX NEG and at 5 mg/L to the MIX POS.

DETERMINATION OF DEAD TIME
- Method: The dead time t0 was measured by using a non-retained organic compound (sodium nitrate) which was present in the reference items mixes and in the test item solution.

REFERENCE SUBSTANCES
The reference items that were used in this study were chosen after non-GLP preliminary determination.
- Identity:
Phenol (CAS 108-95-2; purity 99.9%; log Koc 1.32)
Atrazine (CAS 1912-24-9; purity 99%; log Koc 1.81)
Isoproturon (CAS 34123-59-6; purity 99.8%; log Koc 1.86)
Triadimenol (CAS 55219-65-3; purity 98.2%; log Koc 2.40)
Triazoxide (CAS 72459-58-6; purity 99.4%; log Koc 2.44)
Linuron (CAS 330-55-2; purity 99.7%; log Koc 2.59)
Endosulfan alcohol (CAS 2157-19-9; purity 99%; log Koc 3.02)
Pyrazophos (CAS 13457-18-6; purity 99.7%; log Koc 3.65)
Diclofop-methyl (CAS 51338-27-3; purity 99.63%; log Koc 4.20)
DDT (CAS 50-29-3; purity 99.4%; log Koc 5.63)

DETERMINATION OF RETENTION TIMES
Two vials were filled for the two reference items mixes, and two others with the test item solution. The items were injected in the following order:
- MIX POS (x 1)
- MIX NEG (x 1)
- test item (x 2)
- MIX POS (x 1)
- MIX NEG (x 1)
A blank solution was injected in order to check that no signal was observed from contamination or interfering compounds.

REPETITIONS
- Number of determinations: 2

EVALUATION
- Calculation of capacity factors k': The capacity factor k for every peak was calculated using the following equation: k = (tR - t0) / t0 where tR is the HPLC retention time of test or reference substance (min); t0 is the HPLC dead-time = retention time of sodium nitrate (min).
- Calibration log KOC versus log k: The calibration function was drawn up from the two measurements of the capacity factors of each reference item. The corresponding logarithms of the adsorption coefficient, log KOC, were plotted as a function of log k. A linear regression was performed and a calibration function was obtained.
- Determination of the log Koc value: The partition coefficient of the test substance was obtained for the two measurements by interpolation of the calculated capacity factor on the calibration graph. Mean and standard deviations of the log KOC were calculated.

Key result

Sample No.:

#1

Type:

log Koc

Value:

3.67 dimensionless

pH:

6.71

Temp.:

30 °C

Key result

Sample No.:

#2

Type:

log Koc

Value:

3.67 dimensionless

pH:

6.71

Temp.:

30 °C

Details on results (HPLC method):

- Retention times of reference substances used for calibration: See table 5.4.1/2 in "Any other information on results incl. tables". The sodium nitrate, used for the determination of the dead time, was only detected with the electrospray negative mode. The retention time of this compound was to 1.13 min for the two injections with this ionisation mode. Therefore, the dead time considered for this study is to 1.13 min.
- Details of fitted regression line (log k' vs. log Koc): The table 5.4.1/3 in "Any other information on results incl. tables" presents for each reference item their log KOC, the calculated retention factor for the two injections (k1 and k2), the mean of k1 and k2 (k mean), and log (k mean). The logarithms of the adsorption coefficients, log KOC, are plotted as a function of the mean capacity factors, log k. The linear regression obtained with the reference items is presented in the attached graph with the representation of the confidence interval (in orange) calculated with a 98 % confidence level. The determination coefficient R² observed is 0.9739 and the correlation coefficient R is 0.9869. According to the guideline, a typical correlation coefficient for the relationship between log KOC and log k for a set of test substances is around 0.95. Therefore, the obtained coefficients are satisfactory.
The equation of the line issued from this linear regression will be used for the test item log KOC calculation.
- Average retention data for test substance: The retention times obtained are presented in the table 5.4.1/4 in "Any other information on results incl. tables". The calculated values (k, log k and log Koc) for the test item are presented in the table 5.4.1/5 in "Any other information on results incl. tables". log KOC is calculated using the regression equation obtained from the reference items: log Koc = 4.1871 x log k + 0.6980.

Table 5.4.1/2: Retention times of the reference items

Compound	tR 1 min)	tR 2(min)	ΔtR(min)	tR mean (min)	ΔtR/ tRmean	Equilibrium ?
phenol	2.73	2.72	0.01	2.73	0.37%	YES
atrazine	3.02	3.02	0.00	3.02	0.00%	YES
isoproturon	3.21	3.16	0.05	3.19	1.57%	YES
triadimenol	3.93	3.88	0.05	3.91	1.28%	YES
triazoxide	3.93	3.90	0.03	3.92	0.77%	YES
linuron	4.95	4.87	0.08	4.91	1.63%	YES
endosulfan alcohol	6.24	6.12	0.12	6.18	1.94%	YES
pyrazophos	7.16	6.97	0.19	7.07	2.69%	YES
diclofop methyl	8.43	8.15	0.28	8.29	3.38%	YES
DDT	16.52	15.94	0.58	16.23	3.57%	YES

with tR: retention time; ΔtR: absolute value of the retention times difference

Table 5.4.1/3: log Koc and log k values for the reference items

Compound	log KOC	k1	k2	k mean	log (k mean)
phenol	1.32	1.42	1.41	1.42	0.15
atrazine	1.81	1.67	1.67	1.67	0.22
isoproturon	1.86	1.84	1.80	1.82	0.26
triadimenol	2.40	2.48	2.43	2.46	0.39
triazoxide	2.44	2.48	2.45	2.47	0.39
linuron	2.59	3.38	3.31	3.35	0.53
endosulfan alcohol	3.02	4.52	4.42	4.47	0.65
pyrazophos	3.65	5.34	5.17	5.26	0.72
diclofop methyl	4.20	6.46	6.21	6.34	0.80
DDT	5.63	13.62	13.11	13.37	1.13

Table 5.4.1/4: Retention times of the test item

	Injection 1 tR (min)	Injection 2 tR (min)	tR mean (min)	Relative standard deviation
Dead time t0	1.13	1.13	1.13	0.0%
Test item	6.91	6.88	6.90	0.3%

Table 5.4.1/5: Calculated values of log Koc for the test item

	Test item
Measurement No.	k	log k	log KOC
1	5.12	0.71	3.67
2	5.09	0.71	3.67
log KOCmean			3.67
Standard deviation			0.000

Validity criteria fulfilled:

yes

Conclusions:

Using the HPLC method, the following adsorption coefficient was determined for the test item: log KOC = 3.67.
This value is the mean of two independent measurements.

Executive summary:

A study was performed to estimate the Adsorption Coefficient on Soil and Sewage Sludge of the test item using HPLC (High Performance Liquid Chromatography). The method followed was designed to be compliant with the OECD Guideline for Testing of Chemicals No. 121 and EU method C.19.

The adsorption coefficient (KOC) is defined as the ratio between the concentration of the substance in the soil/sludge and the concentration of the substance in the aqueous phase at adsorption equilibrium. The adsorption coefficient, being the quotient of two concentrations, is dimensionless and is usually given in the form of its logarithm to base ten (log KOC).

HPLC is performed on analytical columns packed with a commercially available cyanopropyl solid phase containing lipophilic and polar moieties. A moderately polar stationary phase based on a silica matrix is used. 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 the stationary phase having polar and non-polar sites allows for interaction of polar and non-polar groups of a molecule with 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 adsorption coefficient on organic matter to be established.

Ten reference items with different retention times were used to produce the calibration curve. They were chosen in relation with the expected retention time of the test item. For each reference item, two determinations were performed. The capacity factor k was calculated from the retention time of sodium nitrate (dead time) and the retention time of the respective reference item. A calibration function (log KOC versus log k, linear fit) was determined based on literature values of KOC for the reference items.

The following adsorption coefficient was determined for the test item: log KOC = 3.67. This value is the mean of two independent measurements.

Description of key information

OECD Guideline 121, EU Method C.19, GLP, key study, validity 1:

Koc = 4 677; log Koc = 3.67.

Slightly mobility in soils (according to P.J. McCall et al., 1981).

Key value for chemical safety assessment

Koc at 20 °C:

4 677

Additional information

To assess the adsorption potential of the registered substance, one key experimental study is available.

This study (LPL, 2019) was performed on the registered substance according to OECD Guideline 121 and EU Method C.19 (HPLC screening method) under GLP. According to this study, the adsorption coefficient of the registered substance was determined at 4 677, corresponding to a log Koc value at 3.67. The result indicates that the substance is slightly mobile in soils (according to P.J. McCall et al., 1981¹).

¹McCall 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.

Mobility classification scheme:

Range of Koc	Mobility class
0 - 50	Very high
50 - 150	High
150 - 500	Medium
500 - 2000	Low
2000 - 5000	Slightly
> 5000	Immobile