2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium methyl sulphate

Adsorption / desorption

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

adsorption / desorption: screening

Remarks:

Information from Zahn-Wellens test

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24.07.1991-21.08.1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 302 B

Version / remarks:

Version: 1981.
The Zahn-Wellens test is a test for biodegradability

Deviations:

no

GLP compliance:

yes

Type of method:

other: Screening study on inherent biodegradability

Media:

sewage sludge

Radiolabelling:

no

Test temperature:

22+/-3 °C

Sample No.:

#1

Type:

other: % degradation (DOC removal)

Remarks:

65.5%

Value:

65.5 other: % removal after first 3 h of experiment

Matrix:

150 mg/L test

Remarks on result:

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

Sample No.:

#2

Type:

other: % degradation (DOC removal)

Value:

90 other: % removal after first 3 h of experiment

Matrix:

50 mg/L test

Remarks on result:

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

Test substance (322 mg/L)

Elimination measured as DOC (150 mg/l) after 28 days corrected by the blank control is: 92.9 % (average of 2 tests running in parallel)

Adsorption after 3 hours: 65.5 %

Elimination measured as DOC (50 mg/l) after 28 days corrected by the blank control is: 100.0 % (average of 2 tests running in parallel)

Adsorption after 3 hours: 90.0 %

Reference substance

Biodegradation corrected by the blank control and measured as DOC (mg/l) is: 100 % (16th day)

Validity criteria fulfilled:

not applicable

Conclusions:

The high elimination within the first 3 hours of the experiments 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 150 mg DOC/L. Biodegradation was followed by measurement of DOC removal from the test vessels. In the reference item 1.8 % elimination of DOC could be observed after 3 h. After 7 d 87 % DOC have been removed.

In contrary where the test substance was added (50.0 DOC mg/L) 90.0 % DOC was eliminated after 3 h, and for the second test (150 mg DOC/L) 65.5 % elimination was reached after 3 h, indicating 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.

Reference 2

Endpoint:

adsorption / desorption: screening

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Unknown

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: Activated Sludge Simulation Test OECD 303 A mod.

Deviations:

yes

Remarks:

no control units included

Principles of method if other than guideline:

Assessment of elimination of test substance in laboratory scale activated sludge units (Husmann).
Mean retention time in activated sludge units: 6h
Total test duration: 31 days

GLP compliance:

not specified

Type of method:

other: Within the activated sludge unit e.g.: HPLC, TOC

Media:

sewage sludge

Radiolabelling:

no

Test temperature:

not reported

Remarks on result:

not measured/tested

Remarks:

The adsorption coefficient was not measured.

Remarks on result:

not measured/tested

Remarks:

The Partition coefficient was not measured.

Validity criteria fulfilled:

not applicable

Conclusions:

It was found that approx. 98 % of the recovered test item adsorbed to the activated sludge.

Executive summary:

The dye was over 90% eliminated over the entire test period (95%, average) from the continuously added solution (= difference of influent and effluent). The test item is a cationic compound that adsorbs quickly and strongly to surfaces, especially organic material such as activated sludge.

By means of a substance-specific analysis, it was possible to determine the dye conversion rate of the total assets including the activated sludge. The samples were taken with a time spacing of 1-3 days. From the concentrations analysed, balance sheets were prepared containing the individual time sections from sampling to sampling and the cumulatively calculated for the entire experimental period.

It was found that approx. 98 % of the recovered dye adsorbed to the activated sludge.

Reference 3

Endpoint:

adsorption / desorption: screening

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the attached report in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Media:

sewage sludge

Sample No.:

#1

Type:

other: % degradation (DOC removal)

Value:

65.5 other: % removal within the first 3 h of the experiment

Matrix:

150 mg/L test

Remarks on result:

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

Sample No.:

#2

Type:

other: % degradation (DOC removal)

Value:

90 other: % removal within the first 3 h of the experiment

Matrix:

50 mg/L test

Remarks on result:

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

Validity criteria fulfilled:

yes

Conclusions:

The high elimination within the first 3 hours of the experiments 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. In the reference item 1.8 % elimination of DOC could be observed after 3 h. After 7 d 87 % DOC have been removed.

In contrary where the test substance was added (50.0 DOC mg/L) 90.0 % DOC was eliminated after 3 h, and for the second test (150 mg DOC/L) 65.5 % elimination was reached after 3 h, indicating 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.

This information is used in a read-across approach in the assessment of the target substance.

For details and justification of read-across please refer to the attached report in section 13 of IUCLID.

Reference 4

Endpoint:

adsorption / desorption: screening

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the attached report in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Remarks on result:

not measured/tested

Remarks:

The adsorption coefficient was not measured.

Remarks on result:

not measured/tested

Remarks:

The Partition coefficient was not measured.

Validity criteria fulfilled:

not applicable

Conclusions:

It was found that approx. 98 % of the recovered test item adsorbed to the activated sludge.

Executive summary:

The dye was over 90% eliminated over the entire test period (95%, average) from the continuously added solution (= difference of influent and effluent). The test item is a cationic compound that adsorbs quickly and strongly to surfaces, especially organic material such as activated sludge.

By means of a substance-specific analysis, it was possible to determine the dye conversion rate of the total assets including the activated sludge. The samples were taken with a time spacing of 1-3 days. From the concentrations analysed, balance sheets were prepared containing the individual time sections from sampling to sampling and the cumulatively calculated for the entire experimental period.

It was found that approx. 98 % of the recovered dye adsorbed to the activated sludge.

This information is used in a read-across approach in the assessment of the target substance.

For details and justification of read-across please refer to the attached report in section 13 of IUCLID.

Reference 5

Endpoint:

adsorption / desorption, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

July 26, 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1.SOFTWARE: EpiSuite v4.1
2.MODEL: KOCWIN v2.00
3.SMILES: COc1cc2sc(n(C)(OS(=O)(=O)OC)c2cc1)N=Nc3ccc(cc3)N(CC)CCO

4.SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: Adsorption / Desportion (log Koc)
- Unambiguous algorithm: Koc estimation by first-order Molecular Connectivity Index methodology (MCI)
- Defined domain of applicability:
Explanations are derived from the on-line KOCWINTM User’s Guide (v2.00). The original KOCWIN used Molecular Connectivity Indices (MCI) and a series of group contributing factors to predict Koc (Meylan et al., 1990). This group contribution method was shown to outperform traditional estimation methods based on octanol/water partition coefficients and water solubility. Since the introduction of the original PCKOC program in 1992, the number of available experimental Koc values has grown significantly. Using an expanded experimental dataset and the original PCKOC methodology, the QSAR equations were re-regressed to derive updated coefficient values. In addition, several new group contribution factors (correction factors) were added to improve estimation accuracy. Also, the updated KOCWIN program includes a separate Koc estimate based upon Log Kow (rather than MCI).
KOCWIN (version 2) implemented in EPI SuiteTM (US EPA, 2017) was used to estimate the adsorption coefficient of Basic Blue 41 methyl sulphate. The program uses two separate estimation methodologies:
1) Estimation using first-order Molecular Connectivity Index (MCI)
2) Estimation using log Kow (octanol-water partition coefficient)
However, the estimation using log Kow is not relevant for Basic Blue 41 methyl sulphate as the log Kow is considered a poor descriptor for the adsorption behaviour of this cationic and surface active substance.
The KOCWIN program is based on experimental Koc values from 674 compounds, eventually divided into a training set of 516 compounds (69 non-polar organics and 447 polar organics) and a validation set of 158 compounds.

- Appropriate measures of goodness-of-fit and robustness and predictivity:
The previous KOCWIN program (PCKOCWIN version 1.66, EPI Suite v3.20) was trained with a total dataset of 189 compounds as described in Meylan et al. (1992). The training and validation statistics for the previous version were:
- Training Non-polar: number = 64; r² = 0.956; std dev = 0.267; avg dev = 0.211;
- Training (with corrections) = 125; r² = 0.918; std dev = 0.210; avg dev = 0.168;
The new MCI methodology uses 516 compounds in the training sets (compared to 189 compounds in the older MCI regression). Using the new 516 compound dataset and 158 compound validation set, the following tables list the estimation accuracy of the new KOCWIN program versus the previous version.  In this comparison, the older version was used to predict log Koc of the updated experimental dataset to illustrate the improvement in estimation accuracy of the new KOCWIN program:
- Training Data [non-polar (no corrections)]:
* New KOCWIN: number = 69; r² = 0.967; std dev = 0.247; avg dev = 0.199;
* Old PCKOC = 69; r² = 0.967; std dev = 0.248; avg dev = 0.200;
- Training Data [non-polar (with correction factors)]:
* New KOCWIN: number = 447; r² = 0.900; std dev = 0.340; avg dev = 0.273;
* Old PCKOC = 477; r² = 0.781; std dev = 0.505; avg dev = 0.365;
- Validation Dataset:
* New KOCWIN: number = 158; r² = 0.850; std dev = 0.583; avg dev = 0.459;
* Old PCKOC = 158; r² = 0.739; std dev = 0.779; avg dev = 0.558;
The basic Non-Polar equation changed very little from the old version to the new version, and the statistics for the Non-Polar Training compounds confirm the close similarity. The coefficients have changed slightly and 7 new correction factors have been added to the new version which is the major reason the accuracy has been improved.

- Mechanistic interpretation:
The Soil Adsorption Coefficient Programm (KOCWIN) estimates the soil adsorption coefficient (Koc) of organic compounds. Koc can be defined as "the ratio of the amount of chemical adsorbed per unit weight of organic carbon (oc) in the soil od sediment to the concentration of the chemical in solution at equilibrium (Lyman, 1990): it is represented by the following equation (Lyman, 1990): Koc = (µg adsorbed organic carbon) / (µg/ml solution).
Koc provides an indication of the extent to which a chemical partitions between solid and solution phases in soil, or between water and sediment in aquatic ecosystems. Traditional estimation methods rely upon the octanol/water partition coefficient or related parameters, but the first-order molecular connectivity index (MCI) has been used successfully to predict Koc values for hydrophobic organic compounds ( Sabljic, 1984, 1987; Bahnick and Doucette, 1988). The original KOCWIN program (PCKOC) used MCI and a series of group contribution factors to predict Koc ( Meylan et al., 1992). This group contribution method was shown to outperform traditional estimation methods based on octanol/water partition coefficients and water solubility.
Since the introduction of the original PCKOC program in 1992, the number of available experimental Koc values has grown significantly. Using an expanded experimental dataset and the original PCKOC methodology, the QSAR equations were re-regressed to derive updated coefficient values.  In addition, several new group contribution factors (correction factors) were added to improve estimation accuracy. Also, the updated KOCWIN program includes a separate Koc estimate based upon Log Kow (rather than MCI).  A brief description of the estimation methodology and accuracy is presented in the Methodology section and Accuracy section of this help file.

KOCWIN requires only a chemical structure to make these predictions.  Structures are entered into KOCWIN by SMILES (Simplified Molecular Input Line Entry System) notations.  A discussion of the encoding rules for SMILES notation can be found in the document " A Brief Description of SMILES Notation".  
The following journal article explains the MCI prediction methodology and its use:
Meylan, W., P.H. Howard and R.S. Boethling, "Molecular Topology/Fragment Contribution Method for Predicting Soil Sorption Coefficients", Environ. Sci. Technol. 26: 1560-7 (1992).

The soil adsorption coefficient of an organic substance is highly dependent on the molecular structure. To be more specific, hydrophobic (non-polar) and hydrophilic (polar) functional groups present in the respective molecule are responsible for adsoprtion / desorption behaviour between solid and solution phases in soil, or between water and sediment in aquatic ecosystems. Therefore, partitioning of the chemical structure and estimating the Koc based on defined values for the organic compound is a reasonable approach.

5. APPLICABILITY DOMAIN
-Descriptor domain: Currently there is no universally accepted definition of model domain. However, users may wish to consider the possibility that log Koc estimates are less accurate for compounds outside the MW range of the training set compounds, and/or that have more instances of a given fragment than the maximum for all training set compounds. It is also possible that a compound may have a functional group(s) or other structural features not represented in the training set, and for which no fragment coefficient or correction factor was developed. These points should be taken into consideration when interpreting model results. Basic Blue 41
i. does have a molecular weight of 482.6 g/mol which is inside the molecular weight range of the training set compounds (ca. 32 - 665 g/mol);
ii. does not have more instances of a given fragment than the maximum for all training set compounds;
iii. does have one structural group, the sulphate (-SO4-) group, which is not available in the training set but considered by one miscellaneous S(=O) group in estimation result;
iv. does have one additional structural group, the thiol (-SH-) group which is not represented in the training set and thus not considered in estimation result;
v. metabolic domain: not relevant.

- Similarity with analogues in the training set: Structural analogues were not identified in the training or validation data set.

6. ADEQUACY OF THE RESULT
Basic Blue 41 methyl sulphate has a MW of 482.57 and thus within the MW range of the training set (minimum MW: 32.04, maximum MW: 665.02, average MW: 224.4), and does not exceed the maximum number of instances of a given fragment from the training set. Despite having additional structural groups not considered in the training set, it is concluded that the log Koc prediction by MCI methodology for Basic Blue 41 methyl sulfate is sufficiently accurate to be used in a weight-of-evidence approach in the assessment of the adsorptive behaviour of the substance. The result of the QSAR prediction is further supported by the experimentally determined adsorption / desorption behaviour of the substance.

Qualifier:

no guideline required

Principles of method if other than guideline:

QSAR calculation

GLP compliance:

no

Type of method:

other: QSAR calculation

Type:

Koc

Value:

4 599 L/kg

Remarks on result:

other: pH and temperature not reported in the QSAR prediction.

Type:

log Koc

Value:

3.663 dimensionless

Remarks on result:

other: pH and temperature not reported in the QSAR prediction.

Validity criteria fulfilled:

not specified

Conclusions:

The adsorption behaviour of Basic Blue 41 methyl sulfate estimated by EpiSuite v4.1 / KOCWIN v2.00 using the Molecular Connectivity Index (MCI) methodology resulted in a log Koc value of approximately 3.7.

Executive summary:

The adsorption behaviour of Basic Blue 41 methyl sulfate estimated by EpiSuite v4.1 / KOCWIN v2.00 using the Molecular Connectivity Index (MCI) methodology resulted in a log Koc value of approximately 3.7. The estimated log Koc value for Basic Blue 41 methyl sulphate is high and suggests a high adsorption behaviour of the substance to solid phases in soil and sediment in aquatic ecosystems.

Basic Blue 41 methyl sulfate has a molecular weight (MW) of 482.57 g/mol and is thus within the molecular weight range of the training set (minimum MW: 32.04 g/mol, maximum MW: 665.02 g/mol, average MW: 224.4 g/mol), and does not exceed the maximum number of instances of a given fragment from the training set. Despite having additional structural groups not considered in the training set, it is concluded that the log Koc prediction by MCI methodology for Basic Blue 41 methyl sulfate is sufficiently accurate to be used in a weight-of-evidence approach in the assessment of the adsorptive behaviour of the substance.

The result of the QSAR prediction is further supported by the experimentally determined adsorption behaviour of the substance.

Reference 6

Endpoint:

adsorption / desorption: screening

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no data

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))

Version / remarks:

2001

Deviations:

yes

Remarks:

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

GLP compliance:

no

Remarks:

Expert statement

Type of method:

HPLC estimation method

Media:

soil/sewage sludge

Specific details on test material used for the study:

Test item: Basic Blue 41 Methylsulfate in acetic acid
Batch No. GU33022

Radiolabelling:

no

Test temperature:

Column temperature 20 - 25 ± 1°C

Details on study design: HPLC method:

Test system:
Analytical HPLC column (based on silica matrix) packed with a cyanopropyl solid phase containing non-polar and polar moieties coupled to DAD. Methanol : borate buffer pH 9 (75 : 25 v/v), methanol : phosphate buffer pH 7 (75 : 25 v/v) and methanol : citrate buffer pH 4 (60 : 40 v/v) were used as mobile phases.

Reasons for the selection:
The selection of the test system was based on the guidelines. HPLC column 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.

HPLC system: HPLC AGILENT 1100 consisting of:
- Auto sampler G1329A
- Auto sampler thermostat G1330B
- Pump G1312A
- Degasser G1322A
- Column oven G1316A
- Detector G1315B
- Software Empower 2 (Build No: 2154), WATERS

Column: YMC-Pack Cyano, 250 x 4.0 mm, YMC EUROPE GMBH
Mobile phase:
pH 9: Methanol : borate buffer 25 mM (75 : 25 v/v)
pH 7: Methanol : phosphate buffer 25 mM (75 : 25 v/v)
pH 4: Methanol : citrate buffer 25 mM (60 : 40 v/v)

Flowrate: 1.0 ml/min

Type:

log Koc

Value:

2.6 dimensionless

pH:

4

Matrix:

soil

Remarks on result:

other: Peak 1

Type:

log Koc

Value:

3.1 dimensionless

pH:

4

Matrix:

soil

Remarks on result:

other: Peak 2

Type:

log Koc

Value:

2.5 dimensionless

pH:

4

Matrix:

sewage sludge

Remarks on result:

other: Peak 1

Type:

log Koc

Value:

3.1 dimensionless

pH:

4

Matrix:

sewage sludge

Remarks on result:

other: Peak 2

Type:

log Koc

pH:

7

Matrix:

soil & sewage sludge

Remarks on result:

not determinable because of methodological limitations

Type:

log Koc

pH:

9

Matrix:

soil & sewage sludge

Remarks on result:

not determinable because of methodological limitations

Non-GLP measurements were performed with mobile phases consisting of methanol and aqueous buffer solutions of pH 4, pH 7 and pH 9. The chromatographic capacity factor k' of the test item was found to be influenced by the pH value. The retention time (corresponding to the capacity factor) of the test item increases at lower pH values. At neutral or basic pH, the test item has a much higher retention time than phenanthrene and 4,4'-DDT (Table 1). 4,4'-DDT is the reference compound given in the guideline with the highest log Koc value for soil, phenanthrene the reference compound with the highest log Koc value for sewage sludge. A determination of the log Koc would only be possible by extrapolation, which provides significant inaccuracy for the study endpoint. At pH 4, k' of the test item is lower than that of the reference compounds but the chromatographic signal splits into two peaks. A calculation of log Koc values is possible and shown below in Table 2.

Table 1: Determined Retention Times of the Reference Compounds and the Test Item at pH 7 and pH 9

Reference item	Retention time [min]
	pH 7	pH 9
Sodium nitrate (deadtime)	1.971	2.158
4-Nitrobenzamide	3.319	3.713
Monuron	n.d.	3.788
Phenylbenzoate	3.893	4.265
Phenanthrene	4.601	5.081
4,4'-DDT	5.610	6.073
Test Item	16.361	22.097

Table 2: Capacity Factors (k') of the Reference Items for Soil and Sewage Sludge at pH 4

Reference item	log Koc, soil	log Koc, sewage sludge	Calibration
			tR [min]	k'	log k'
4-Nitrobenzamide	1.93	1.52	4.236	0.289	-0.540
Monuron	2.55	2.45	5.845	0.778	-0.109
Phenylbenzoate	2.87	3.26	6.615	1.01	0.00538
Phenanthrene	4.09	4.35	9.819	1.99	0.298
4.4'-DDT	5.63	-	20.689	5.29	0.724

Since the retention behavior of the test item is significantly influenced by the pH value of the mobile phase and no adsorption coefficient can be calculated at neutral conditions, the guidelines are not applicable. Only at pH 4, a log Koc value was calculated for two merging peaks (see Table 3). It should be considered that such acidic pH values are not relevant for the ecotoxicological risk assessment. The substance can be considered highly adsorptive to soil and sewage sludge at neutral and basic pH.

Table 3: Determined log Koc (non-GLP) for the Test Item at pH 4

	log Koc,soil	log Koc,sewage sludge
Test Item Basic Blue 41 Methylsulfate, Peak 1	2.6 ± 0.2 1.9 to 3.2 (95 % Cl)	2.5 ± 0.2 1.8 to 3.2 (95 % Cl)
Test Item Basic Blue 41 Methylsulfate, Peak 2	3.1 ± 0.2 2.6 to 3.7 (95 % Cl)	3.1 ± 0.2 2.4 to 3.8(95 % Cl)

Validity criteria fulfilled:

not applicable

Conclusions:

Since the retention behaviour of the test item is significantly influenced by the pH value of the mobile phase and no adsorption coefficient can be calculated at neutral conditions, the guidelines are not applicable. Only at pH 4, a log Koc value was calculated for two merging peaks. It should be considered that such acidic pH values are not relevant for the ecotoxicological risk assessment. The substance can be considered highly adsorptive to soil and sewage sludge at neutral and basic pH.

Executive summary:

Pre-tests with the test item were conducted prior to a main study (OECD 121) at pH 4, 7 and 9 in buffered media as the test item is considered ionic. During these pre-tests, considerable technical difficulties were encountered:

The retention time of the test substance at neutral and basic pH 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 would only be possible by extrapolation, which would be associated with significant inaccuracy for the endpoint estimate (log Koc).

The pH value had a strong influence on the chromatographic behaviour of the test item. The retention time of the test substance increased with increasing pH (which is equivalent to increasing adsorption with increasing pH). At pH 7 and 9 the retention time of the test item was 16.361 and 22.097 minutes, respectively. The test item considerably exceeded the retention time of the reference substances phenanthrene (pH 7: 4.601 min, pH 9: 5.081 min) and 4,4’-DDT (pH 7: 5.610, pH 9: 6.073 min) and was thus outside of the validated range of OECD 121. Only at pH 4, the retention time of the test substance was within the validated range of the test method. However, the chromatographic signal splits into two peaks (peak 1: 5.338 min, peak 2: 6.385 min).The calculated log Koc values of Basic Blue 41 methyl sulphate in soil and sewage sludge at pH 4 are depicted in the table below.

Determined log Koc (non-GLP) of the test substance at pH 4

Basic Blue 41 methyl sulphate	Log Koc, soil	Log Koc, sewage sludge
Peak 1	2.6 ± 0.2 1.9–3.2 (95 % C.I.)	2.5 ± 0.2 1.8–3.2 (95 % C.I.)
Peak 2	3.1 ± 0.2 2.6–3.7 (95 % C.I.)	3.1 ± 0.2 2.4–3.8 (95 % C.I.)

 

Thus, OECD 121 is considered unsuitable for the measurement of the log Koc of the test item at pH 7 and 9.

Description of key information

The test item is a surface-active substance with a low log Kow (0.3) and high 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 within the scope of an OECD 121 screening study, considerable technical difficulties were encountered. Measurement of the log Koc at pH 7 and 9 is not possible, but an approximation of mean log Koc values of 2.6 (peak 1) / 3.1 (peak 2) and 2.5 (peak 1) / 3.1 (peak 2) at pH 4 in soil and sewage sludge, respectively, can be provided. It should be considered that such acidic pH values are not relevant for the ecotoxicological risk assessment.

Additional information from tests according to OECD 302 B (Zahn-Wellens Test) and OECD 303 A (activated sludge units) demonstrate rapid removal of the test substance from the test solution: in the OECD 302 B test values of 90 % and 65.5 % elimination within 3 h after start of the test in the 50 mg TOC/L and 150 mg TOC/L test solution, respectively, while approximately 98 % of the substance was found to be adsorbed to activated sludge.

Based on the experimental results described above and considering the charged moieties within the compound, it can be concluded that the test item has a high adsorption potential in soil and sewage sludge. This assumption is further supported by the QSAR estimated log Koc of ca. 3.7 (MCI method).

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 the test item can be expected to be significantly higher than the estimates for pH 4 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

[LogKoc: 6.0]