[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl](2-hydroxypropyl)dimethylammonium chloride

Adsorption / desorption

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

adsorption / desorption: screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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

Deviations:

yes

Remarks:

Test restricted to preliminary investigations due to non-applicability of the test guideline.

GLP compliance:

yes

Remarks:

However, the preliminary tests are not conducted under GLP

Type of method:

HPLC estimation method

Media:

soil/sewage sludge

Test temperature:

Column temperature 20 - 25 ± 1°C

Details on study design: HPLC method:

EQUIPMENT
- Analytical HPLC column (based on silica matrix) packed with a cyanopropyl solid phase containing non-polar and polar moieties coupled to DAD. Column: YMC-Pack Cyano, 250 x 4.0 mm, YMC EUROPE GMBH. Column temperature: 20 - 25 ± 1°C. Flow: 1.0 ml/min
- Detection system: DAD-Detector, ACQUITY UPLC Photodiode Array Detector, WATERS
- Autosampler: Acquity UPLC, WATERS
- Binary Solvent Manager: Acquity UPLC, WATERS
- Column Manager: Acquity UPLC, WATERS
- Software: Empower 2 (Build No: 2154), WATERS

MOBILE PHASES
- Type:
pH 9: Methanol : borate buffer 25mM (70 : 30 v/v)
pH 7: Methanol : phosphate buffer 25 mM (70 : 30 v/v)
pH 4: Methanol : citrate buffer 25mM (65 : 35 v/v)
- Experiments with additives carried out on separate columns: no
- pH: see above
- Solutes for dissolving test and reference substances:

ADDITIONAL EQUIPMENT
- Analytical balances, SARTORIUS
- Positive displacement pipettes, Microman, GILSON
- Piston stroke pipettes, Finnpipette, THERMO SCIENTIFIC

DETERMINATION OF DEAD TIME
- Method: by means of homologous series / by inert substances which are not retained by the column

REFERENCE SUBSTANCES - Identity:
1) Dead Time
- Substance NaNO3, SIGMA-ALDRICH
- Batch No. SZBE1280V
- CAS No. 7631-99-4
- Purity 100.1%
- Expiry date 2019-04-12

2) Reference Items for Adsorption Coefficient on Soil and Sewage Sludge
- No.1 Monuron, SIGMA-ALDRICH
- Batch No. SZBD114XV
- CAS No. 150-68-5
- Purity 99.9% (HPLC area%)
- log Koc. soil 1.99
- log Koc. sewage sludge 1.46
- Expiry date 2018-04-24

- No. 2 2,5-Dichloroaniline, ALDRICH
- Batch No. STBB4341V
- CAS No. 95-82-9
- Purity 99.8% (GC area%)
- log Koc. soil 2.55
- log Koc. sewage sludge 2.45
- Expiry date 2021-10-18

- No. 3 Linuron, CHEMSERVICE
- Batch No. 1679500
- CAS No. 330-55-2
- Purity 98.1 % (HPLC %)
- log Koc. soil 2.59
- log Koc, sewage sludge 2.43
- Expiry date 2017-01-31

- No. 4 Fenthion, SIGMA-ALDRICH
- Batch No. SZBD170XV
- CAS No. 55-38-9
- Purity 97.5% (HPLC area%)
- log Koc. soil 3.31
- log Koc. sewage sludge 3.75
- Expiry date 2018-06-19

- No. 5 Phenanthrene, SuPELCO
- Batch No. XA13459V
- CAS No. 85-01-8
- Purity 99.0 % (GC-FID)
- log Koc. soil 4.09
- log Koc. sewage sludge 4.35
- Expiry date 2018-03

- No. 6 4,4'-DDT, SIGMA-ALDRICH
- Batch No. SZBC167XV
- CAS No. 50-29-3
- Purity 99.8 % (GC area%)
- log Koc. soil 5.63
- Expiry date 2017-06-15

Type:

log Koc

Value:

> 5.63 dimensionless

pH:

4

Matrix:

soil

Remarks on result:

not determinable because of methodological limitations

Remarks:

estimated value

Type:

log Koc

Value:

> 4.35 dimensionless

pH:

4

Matrix:

sewage sludge

Remarks on result:

not determinable because of methodological limitations

Remarks:

estimated value

Details on results (HPLC method):

- Retention times of reference substances used for calibration:
* Monuron, t = 3.240 min
* 2,5-Dichloroaniline, t = 3.565 min
* Fenthion, t = 4.728 min
* Phenanthrene, t = 4.839 min
- Details of fitted regression line (log k' vs. log Koc):
Soil:
* Slope = 5.400
* Intercept = 3.9980
* r² = 0.8982
* r = 0.9477
Sewage sludge:
* Slope = 5.601
* Intercept = 4.0980
* r² = 0.9724
* r = 0.9861
- Graph of regression line attached: no
- Average retention data for test substance: pH 4: 6.354 min, pH 7: 30.316 min, pH 9: 40.817 min

In non-GLP measurements the test item Basic Red 18.1 Chloride had a much higher retention time than phenanthrene and 4,4'-DDT. 4,4'-DDT is the reference compound given in the guideline with the highest log Koc value for soil, phenantrene the reference compound with the highest highest log Koc value for soil and sewage sludge. A determination of the log Koc value would only be possible by extrapolation, which provides significant inaccuracy for the study endpoint. Since the test item is ionic, buffered test media at pH 4, pH 7 and pH 9 have been used for preliminary investigations. The results are presented in the table 1 below.

Table 1: Determined Retention Times of the Reference Items and Test Item

Reference item	Retention time			Log Koc, soil	Log Koc, sewage sludge
	pH 4	pH 7	pH 9
Sodium nitrate (dead time)	2.40	1.890	1.884	-	-
Monuron	3.240	3.051	n.d.	1.99	1.46
Linuron	n.d.	n.d.	3.531	2.59	2.43
2,5-Dichloroaniline	3.565	3.267	3.343	2.55	2.45
Fenthion	4.728	3.938	4.055	3.31	3.75
Phenanthrene	4.839	4.081	4.254	4.09	4.35
4,4’-DDT	n.d.	n.d.	5.216	5.63	-
Test item	6.354	30.316	40.817	> 5.63	> 4.35

  n.d. = not determined

Since the retention time of the test item is lower at lower pH values, extrapolated values for pH 4 have been calculated (see tabel 2 below).

Table 2: Retention Times, Capacity Factors (k') and Adsorption Coefficient (log Koc) of Basic Red 18:1 Chloride for Soil and Sewage Sludge - pH 4

	Basic Red 18:1 Chloride
	tR[min]	k’	Logk’	Log Koc, soil 95 % CI	Log Koc, sewage sludge 95 % CI
	6.488	1.70	0.230	51.7 ± 0.39 3.457 to 6.872	5.31 ± 0.31 3.995 to 6.620
	6.223	1.59	0.201
Mean	6.356	1.64	0.216

t_R = Retention time; Cl = Confidence interval

 

Validity criteria fulfilled:

not applicable

Conclusions:

OECD guideline 121 is not applicable for the exact determination of the adsorption coefficient (Koc) of the test item on soil and on sewage sludge. The log Koc of the test item is outside the scope of this guideline. Nevertheless, log Koc values have been extrapolated for pH 4. A log Koc of 5.17 was extrapolated for soil and a log Koc of 5.31 for sewage sludge.

Executive summary:

Preliminary investigations (non-GLP) for the determination of the adsorption coefficient Koc of the test item on soil and on sewage sludge using high performance liquid chromatography (HPLC) were performed according to OECD 121 and Council Regulation (EC) No.440/2008, Method C.19.

The results of these experiments showed that this guideline is not applicable for the determination of the adsorption coefficient (Koc) of the test item on soil and on sewage sludge because it has a much higher retention time than phenanthrene and 4,4'-DDT. 4,4'­ DDT is the reference compound given in the guideline with the highest log Koc value for soil, phenantrene the reference compound with the highest log Koc value for soil and sewage sludge. A determination of the log Koc value would only be possible by extrapolation, which provides significant inaccuracy for the study endpoint. Since the test item is ionic, buffered test media at pH 4, pH 7 and pH 9 have been used for preliminary investigations. Since the retention time of the test item is lower at lower pH values, extrapolated values for pH 4 have been calculated. A log Koc of 5.17 was extrapolated for soil and a log Koc of 5.308 for sewage sludge.

 

Reference 2

Endpoint:

adsorption / desorption, other

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Version / remarks:

August 1989

GLP compliance:

no

Type of method:

other: Biodegradation screening study

Media:

sewage sludge

Radiolabelling:

no

Key result

Sample No.:

#1

Type:

other: % degradation (DOC removal)

Remarks:

within first 3h of experiment

Value:

63 other: %

Remarks on result:

other: The high elimination within the first 3 h of the experiment indicates removal through adsorp tion of the test substance. No adsorption control included in the experiment.

Sample No.:

#1

Type:

other: % degradation (DOC removal)

Remarks:

after 7 days of incubation

Value:

87 other: %

Table 1: Test results

Time	Control DOC [mg/L]	Test substance DOC [mg/L]	Test substance – Control DOC [mg/L]	Degradation [%]
0 h	-	-	100	0
3 h	9	45	37	63
7 d	10	23	13	87

Validity criteria fulfilled:

not applicable

Conclusions:

The high elimination within the first 3 h of the experiment indicates removal through adsorption of the test substance. However, no adsorption control was included in the experiment

Executive summary:

The inherent biodegradability of the test substance was investigated in accordance with OECD Guideline 302 B (Zahn-Wellens Test) using non-adapted domestic sludge. The initial test substance concentration
was 100 mg DOC/L. Biodegradation was followed by measurement of DOC removal from the test vessels. 3 % elimination of DOC could be observed after 3 h. After 7 d 87% DOC have been removed.
The high elimination within the first 3 h of the experiment indicates removal through adsorption of the test substance. However, no adsorption control was included in the experiment. This information is used in a weight-of-evidence approach in the assessment of the adsorption potential of the test item.

Description of key information

Basic Red 18:1 Chloride is a surface-active substance with low log Kow (0.3) and good water solubility. Therefore, measured adsorption coefficients at different, environmentally relevant pH values are required for the assessment of the adsorption potential of the substance.

During pre-tests conducted with Basic Red 18:1 Chloride, considerable technical difficulties were encountered. Measurement of the log Koc at pH 7 and 9 is not possible, but an approximation of the log Koc of 5.17 and 5.308 at pH 4 in soil and sewage sludge, respectively, can be provided by estimation.

Supporting information from an OECD 302 B (Zahn-Wellens Test) demonstrates rapid removal of the test substance from the test solution. A value of 63 % elimination within 3 h after start of the test indicates fast removal through adsorption of the test substance.

Based on the experimental results described above and considering the charged moieties within the compound, it can be concluded that Basic Red 18:1 Chloride has a high adsorption potential in soil and sewage sludge.

The predominant pH of surface waters and sewage treatment plants is approximately 7 or sometimes even higher. Soil pH values can vary considerably depending on soil type, spatial (climate) and temporal distribution. Frequency maxima of forest soil and arable land in Germany are in the range of pH 6-7 and 3.6-4.6 (Schwertmann & Fischer, 1982; cited in Scheffer & Schachtschabel, 2002). Although determination of the Koc by the HPLC method at pH 7 and 9 was not possible, the corresponding value of Basic Red 18:1 Acetate can be expected to be significantly higher than the estimates for pH 4 (5.17 and 5.308 for soil and sewage sludge, respectively). In view of the HPLC retention times log Koc values clearly above 6.0 can be anticipated. For modelling distribution in the environment, a high log Koc is truncated at a value of 6.0 by the program EUSES. Therefore, a log Koc of 6 will be used as basis for exposure estimation by means of Chesar/EUSES in a worst-case approach.

Key value for chemical safety assessment

Koc at 20 °C:

1 000 000

Additional information

DESCRIPTION OF RELEVANT SUBSTANCE PROPERTIES
Basic Red 18:1 Chloride is a solid substance which is characterized by a water solubility of 132 (± 19) g/L at 20 °C and pH 6 (OECD 105). The n-octanol/water partition coefficient (log Kow), that was determined in accordance with OECD Guideline 107, is 0.3 at 23 °C and pH 7. Furthermore, Basic Red 18:1 Chloride is a surface-active compound (surface tension < 60 mN/m at 20 °C).

Based on the physicochemical properties, a substance can be expected to have a low potential for adsorption if the log Kow value is ≤ 3. However, this may not hold true for substances with good water solubility in combination with a low octanol-water partition coefficient, particularly ionic and/or surfaceactive compounds. Such substances – like Basic Red 18:1 Chloride – do not necessarily always have a low adsorption potential.

Basic Red 18:1 Chloride is an ionic substance due to negatively and positively charged moieties within the molecule. The basic chemical structure of the substance itself – the acetate disregarded – is cationic. In accordance with the ECHA Guidance R.7a, especially cationic substances tend to adsorb strongly. Therefore, a measured adsorption coefficient is usually needed to verify any pH-dependence of substance adsorption. Besides, as Basic Red 18:1 Chloride is a surface-active substance, the log Kow value is considered a poor predictor of adsorption. Thus, measured adsorption coefficients at different, environmentally relevant pH values are required.

OVERVIEW OF EXPERIMENTAL RESULTS
1. OECD Guideline 121 – estimation of the adsorption coefficient (Koc) by means of HPLC (see Landsberg, Dr. C., 2016)
Due to the substance properties described in section 2, a study in accordance with OECD Guideline 121 was commissioned to further investigate the adsorption potential of the structural analogue Basic Red 18:1 Chloride on soil and sewage sludge.

Pre-tests with Basic Red 18:1 Chloride were conducted prior to a main study at pH 4, 7 and 9 in buffered media as the test item is considered to be ionic (Landsberg, 2016). During these pre-tests, considerable technical difficulties were encountered:

The retention time of the test substance Basic Red 18:1 Chloride was much higher compared to those of the reference compounds phenanthrene and 4,4'-DDT. 4,4'-DDT is the reference compound given in the guideline with the highest log Koc value for soil, while phenantrene is the reference compound with the highest log Koc value for soil and sewage sludge. Thus, an estimation of the log Koc value of the test substance Basic Red 18:1 Chloride would only be possible by extrapolation, which provides significant inaccuracy for the study endpoint.

The pH value had a strong influence on the chromatographic behaviour of Basic Red 18:1 Chloride. The retention time of the test substance increased with increasing pH (which is equivalent to decreasing adsorption with increasing pH). At pH 7 and 9 the retention time of the test item was ca. 30 and 40 minutes, respectively. Basic Red 18:1 Chloride considerably exceeded the retention time of the reference substances phenanthrene (pH 7: 4.081 min, pH 9: 4.254 min) and 4,4’-DDT (pH 7: n.d., pH 9: 5.216 min) and was thus outside of the validated range given by OECD 121. At pH 4, the retention time of the test substance (6.356 min) was still above the retention time of the reference compound phenanthrene (4.839 min). However, to provide an approximation of the log Koc for the test item, extrapolated values in soil and sewage sludge at pH 4 were calculated at 5.17 and 5.308, respectively. These values are also outside of the validated range of OECD Guideline 121 (log Koc = 1.5–5.0).

Thus, OECD 121 is considered to not be suitable for the measurement of the log Koc of Basic Red 18:1 Chloride.

2. OECD Guideline 302B – Zahn-Wellens Test (see Müller, 1994)
The inherent biodegradability of the structural analogue Basic Red 18:1 Chloride was investigated in accordance with OECD Guideline 302 B (Zahn-Wellens Test) using non-adapted domestic sludge (Müller, 1994). The initial test substance concentration was 100 mg DOC/L. Elimination of the test substance was followed by measurement of DOC removal from the test vessels. 63% elimination of DOC could be observed after 3 h. After 7 days, 87% DOC had been removed.

Although no adsorption control was included in the experiment, the high elimination within the first 3 h of the experiment indicates fast removal through adsorption of the test substance.

(Q)SAR
In accordance with ECHA Guidance R.7 a, the log Koc of organic substances can often be estimated from their octanol-water partition coefficient (Kow), as well as from other properties such as aqueous solubility. Such methods give useful indications on the qualitative/quantitative adsorption coefficient of a substance. Especially for substances that are difficult to test such estimated values are essential.

However, in the case of the ionic Basic Red 18:1 Chloride, the applicability of QSAR models is limited due to the charged moieties within the compound. In addition, the substance is a Quaternary Ammonium Compound (QAC). Adsorption of QACs mainly occurs by an ion-exchange mechanism and depends on cation-exchange capacity of the sorbent and a variety of other parameters (Boethling, 1994). The training set of the estimation program KOCWIN implemented in EPI SuiteTM(US EPA, 2017) for the calculation of the adsorption coefficient does not include any QACs. Therefore, the Koc estimate is outside of the programme's prediction domain.



REFERENCES
Boethling, R. S. 1994. Environmental aspects of cationic surfactants. In J. Cross and E. J. Singer (ed.), Cationic Surfactants: Analytical and Biological Evaluation, vol. 53. Marcel Dekker, Inc., New York, USA.

Müller, G. (1994). Bioabbaubarkeit von Farbstoffen. Testing Laboratory: Bayer AG, Institut für Umweltanalyse und Bewertungen, Geb. W 15, 51368 Leverkusen, Germany. Company owner: DyStar Colours Distribution GmbH. Report Date: 1994-11-08.

Landsberg, C. (2016). Basic Red 18.1 - Chloride Estimation of the Adsorption Coefficient (Koc) on Soil and on Sewage Sludge using High Performance Liquid Chromatography (HPLC) (Statement). Testing laboratory: Noack Laboratorien GmbH, Käthe-Paulus-Straße 1, D-31157 Sarstedt, Germany. Study No. 160803DL / CAH17176 (non-GLP). Company owner: DyStar Colours Distribution GmbH. Report Date: 2016-12-06.

Scheffer & Schachtschabel (2002). Lehrbuch der Bodenkunde. Spektrum Akademischer Verlag, Heidelberg. ISBN: 3-8274-1324-9.

US EPA (2017). Estimation Programs Interface Suite™ for Microsoft® Windows, v 4.11. United States Environmental Protection Agency, Washington, DC, USA.

[LogKoc: 6.0]