Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: screening test, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Well documented journal article utilising monitoring data in a WWTP

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The authors of this publication present the results of a 2 year monitoring programme assessing the removal and degradation of linear alkylbenzene sulphonate from a wastewater treatment plant.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

other: Central active sludge wastewater treatment plant (WWTP) of Torino

Details on inoculum:

The central active sludge wastewater treatment plant (WWTP) of Torino, an industrial town in northern Italy with approximately 1.5 million inhabitants over a metropolitan area of about 315 km2, through which the Po River runs.

Duration of test (contact time):

2 yr

Based on:

other: intrinsic LAS to the WWTP

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

The results reported here refer to two surveys of the Torino WWTP , made in April 1989 and February 1990, that consisted of daily sampling programs. Inlets and outlets of the plant were totally sampled 12 times in April 1989 and eight times in February 1990. Samples were flow-proportional, average daily samplings taken hourly, automatically for waters and manually for sludges. Final dried sludges and sand were single samples taken occa- sionally. The specific measurements of LAS were completed with flow rate measurements of the various streams and with other unspecific measurements.

Reference substance:

not required

Preliminary study:

Not applicable

Parameter:

% degradation (test mat. analysis)

Value:

ca. 80

Remarks on result:

other: LAS monitoring study

Details on results:

The flow trend in the period under examination was fairly constant, with deviation not higher than approximately 8%.
Table 3 shows the unspecific determinations of surfactants and organic material entering and leaving the plant. The average high percentage of decrease of these unspecific parameters - removal of MBAS and PPAS higher than 95% -testifies to the efficient operation of the plant.
Table 4 shows the specific LAS concentrations made by HPLC in influents and effluents. The LAS content agrees with what is known from the literature, with values in milligrams per liter in water phases (approximately 3 mg/L in influents and approximately 0.1 mg/L in effluents after filtration) and in grams per kilogram in SS, sand, and sludges (i.e., 12-14 g/kg in sludges and inlet SS).
Concerning the average distribution values of carbon of the various LAS homologs found in waters, SS, and sludges of the plant In influent and effluent filtered waters, there is about the same distribution with an increase of short-chain (Cloand C11) homologs with respect to the average distribution of commercial LAS. In SS the situation is just the opposite, with an increase of long-chain (C12 and C13) homologs with respect to the distribution of both LAS in water and commercial LAS. The 2-phenyl isomers in the water and adsorbed phases, display distributions similar to those of the corresponding homologs.
Table 5 shows the SPC average concentrations, expressed as equivalent LAS, found in the February 1990 campaign on a few influent and effluent samples. The data are based on HPLC analyses confirmed by GC-MS studies. The average carboxylated alkyl chain length of SPC was shorter in effluent (C6) than in influent (C9). SPCs were found only in the water phase. Tentative SPC measurements on SS and sludges, after careful centrifugation and filtration to separate interstitial waters, exclude any SPC content in these matrices (detection limit 1 mglkg).

Table 3. Unspecific Average Analysis in the WWTP

	MBAS (mg/L)		PPAS (mg/L)		COD (mg/L)		DOC(a) (mg/L)
	Apr 89	Feb 90	Apr 89	Feb 90	Apr 89	Feb 90	Apr 89	Feb 90
Water in	6.2	9.4	2.0	2.4	422	424	19	15
Water out	0.2	0.15	0.05	0.1	50	62	7.9	4.5
% removal	97	98	97	96	88	85	58	70

MBAS = methylene blue-active substance.

PPAS = potassium picrate-active substance.

(a) Filtered through 0.2 um.

Table 4. LAS specific average concentration in teh WWTP

		LAS Concentration
	Analysed	Units	Apr. 89	Feb. 90
Inlet	Water	mg/L	2.55	2.95
	Sand	g/kg	3.4	3.0
	SS	g/kg	14	13
Outlet	Water	mg/L	0.05	0.14
	SS	g/kg	0.35	0.35
	Sludge (a)	g/kg	14	11.5

(a) After digestors

Table 5. Estimate of sulfophenyl carboxylate (SPC)(a) Intermediates in the WWTP, February 1990

Analysed	SPC Concentration (mg/L)
Inlet (water)	~1
Outlet (water)	~1.5

(a) Equivalent LAS measured by HPLC and confirmed by GC-MS.

Validity criteria fulfilled:

not applicable

Interpretation of results:

readily biodegradable

Conclusions:

The main conclusions drawn from the LAS monitoring study on the WWTP of Torino can be summarised as follows:
Primary biodegradation in sewers is at least 12%.
Removal in the WWTP is 98 to 99%.
Primary biodegradation in the WWTP is in the range of 80 to 84 %,of which ultimate biodegradation is approximately 80%.
Based on these data, it can be concluded that the substance can be considered readily biodegradable for the purpose of chemical safety assessment.

Executive summary:

This study presents the results of a 2 year monitoring programme assessing the removal and degradation of linear alkylbenzene sulphonate from a wastewater treatment plant. The results refer to two surveys of the Torino WWTP , made in April 1989 and February 1990, that consisted of daily sampling programs. Inlets and outlets of the plant were totally sampled 12 times in April 1989 and eight times in February 1990. Samples were flow-proportional, average daily samplings taken hourly, automatically for waters and manually for sludges. Final dried sludges and sand were single samples taken occasionally. The specific measurements of LAS were completed with flow rate measurements of the various streams and with other unspecific measurements.

The main conclusions drawn from the LAS monitoring study on the WWTP of Torino can be summarised as follows:

Primary biodegradation in sewers is at least 12%.

Removal in the WWTP is 98 to 99%.

Primary biodegradation in the WWTP is in the range of 80 to 84 %,of which ultimate biodegradation is approximately 80%.

Based on these data, it can be concluded that the substance can be considered readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: screening test, other

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

Reason / purpose for cross-reference:

read-across source

Parameter:

% degradation (test mat. analysis)

Value:

ca. 80

Remarks on result:

other: LAS Monitoring Study

Validity criteria fulfilled:

not applicable

Interpretation of results:

readily biodegradable

Conclusions:

The main conclusions drawn from the monitoring study on the WWTP of Torino can be summarised as follows:
Primary biodegradation in sewers is at least 12%.
Removal in the WWTP is 98 to 99%.
Primary biodegradation in the WWTP is in the range of 80 to 84 %,of which ultimate biodegradation is approximately 80%.
Based on these data, it can be concluded that the substance can be considered readily biodegradable.

Executive summary:

In a one-to-one read-across approach, the substance benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. (data on LAS as part of category approach) is considered appropriate for direct read-across (one-to-one) to benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts (target substance) for the endpoint biodegradation in water: screening tests.

This study presents the results of a 2 year monitoring programme assessing the removal and degradation of linear alkylbenzene sulphonate from a wastewater treatment plant. The results refer to two surveys of the Torino WWTP , made in April 1989 and February 1990, that consisted of daily sampling programs. Inlets and outlets of the plant were totally sampled 12 times in April 1989 and eight times in February 1990. Samples were flow-proportional, average daily samplings taken hourly, automatically for waters and manually for sludges. Final dried sludges and sand were single samples taken occasionally. The specific measurements of LAS were completed with flow rate measurements of the various streams and with other unspecific measurements.

The main conclusions drawn from the LAS monitoring study on the WWTP of Torino can be summarised as follows:

Primary biodegradation in sewers is at least 12%.

Removal in the WWTP is 98 to 99%.

Primary biodegradation in the WWTP is in the range of 80 to 84 %,of which ultimate biodegradation is approximately 80%.

Based on these data, it can be concluded that the substance can be considered readily biodegradable.

A full justification for the read-across approach is presented in IUCLID Section 13.2.

Reference 3

Endpoint:

biodegradation in water: screening test, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

A well documented journal article reporting a simulation study of degradation in activated sludge. The study is considered suitable for the purpose of hazard assessment and exposure assessment.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study monitored the biodegradation of radiolabeled linear alkylbenzene sulfonate in activated sludge by specifically measuring parent, metabolites, and incorporation into biomass as a function of time. Compared to a classic OECD 301 study, the data presented here provides less uncertainty regarding the interpretation of test results and an ability to assess the fate of chemicals, which are biotransformed but not mineralized. Furthermore, the study provides an understanding of potential metabolites and kinetic data more relevant for exposure assessment.

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

Activated sludge mixed liquor was collected from Polk Run Sewage Treatment Plant (STP) on May 15, 1995, and Sycamore Sewage Treatment Plant (STP) on June 25, 1995. Both treatment plants receive mainly domestic wastewater and are located near Cincinnati, OH. The total suspended solids level of both sludges was adjusted to 2500 mg/L by settling.

Duration of test (contact time):

52 h

Initial conc.:

1 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Parameter followed for biodegradation estimation:

CO2 evolution

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

Abiotic controls were prepared by amending sludge samples with 1 g/L mercuric chloride followed by autoclaving for 90 min. The pH of the abiotic sludge was readjusted with 0.1 N NaOH or 0.1 N HCl to that of the fresh sludge (6.5). For each test chemical, 500 mL of bioactive and abiotic sludge was placed into 1-L Erlenmeyer flasks. The LAS working solution (2.0 mL) was slowly added dropwise to the sludge as it was magnetically stirred. The final added concentration was 1 mg/L for the test material. Following addition of the test materials, the sludge was stirred vigorously for 1 min prior to initial sampling. Subsequently, the flasks were incubated on a shaker table (150 rpm) in a controlled temperature room at 20 +/- 2 °C.

Parameter:

% degradation (test mat. analysis)

Value:

76

Sampling time:

52 h

Remarks on result:

other: Combined CO2 evolution and incorporation into biomass

Remarks:

Polk Run STP

Parameter:

% degradation (test mat. analysis)

Value:

66

Sampling time:

52 h

Remarks on result:

other: Combined CO2 evolution and incorporation into biomass

Remarks:

Sycamore STP

Details on results:

Throughout the entire incubation period, more than 90% of the dosed radioactivity in the abiotic controls was recovered in the methanol extracts and chromatographed as parent LAS. Approximately 2% was distributed among the various biomass fractions and represented the nonextracted parent. In the bioactive treatments, significant radioactivity was recovered in both the methanol and aqueous extracts, and TLC analysis indicated the appearance in these extracts of distinct peaks at radiofrequency (rf) values of 0.50 and 0.90 as well as a grouping of unresolved polar peaks with rf values ranging from 0.0 to 0.16.
With both sludges, the disappearance of parent from the test system was rapid, and only 1.8-3.4% remained after 6 h, which corresponds to a typical hydraulic retention time (HRT) in an activated sludge plant.
This disappearance of parent was associated with the appearance of a cluster of poorly resolved polar peaks at rf 0.0- 0.16 and two minor peaks at rf values of 0.5 and 0.9. The polar peaks were the most prominent intermediates and combined accounted for a maximum of 27.5% of the initial radioactivity after 2 h in the Polk Run sludge and 39% of the initial radioactivity after 1 h in the Sycamore sludge. After 6h, they accounted for 9-14 % of the total, and after 52 h, they represented less than 5-11% of the total initial radioactivity. Individually,the two minor peaks at rf values of 0.5 and 0.9 accounted for less than 4% of the starting radioactivity at any time.
With the Polk Run sludge, no lag was observed in the evolution of 14CO2, which reached 41% after 6 h and 54% after 52 h. Incorporation into biomass also began immediately, reached its maximum level after 8 h (33%), and subsequently decreased to 22% after 52 h. In the Sycamore sludge, the evolution of 14CO2 was initially slow during the first hour but reached 38% after 6h and 43% after 52h. Incorporation into biomass was also slow during the first hour but reached its maximum level after 6 h (33%) and decreased to 23% after 52 h.
These data were corrected by subtracting the radioactivity present in each fraction of the abiotic controls. Most of the radioactivity was incorporated into the protein followed by the cell wall and nucleic acid fractions.

Kinetic parameters describing loss of parent, complete biodegradation (mineralisation + incorporation into biomass), and mineralisation of C12 LAS in activated sludge from two STPs (a)

Process	Function	r2	A (%)	K1 (h-1)	K0 (%h)	n	Half-life (h) (b)
Loss of Parent
Polk Run STP	First-order decay	0.935	81.5 +/- 4.9	0.96 +/- 0.16	NA	NA	0.72
Sycamore STP	First-order decay	0.977	94.4 +/- 3.5	1.10 +/- 0.11	NA	NA	0.63
Complete
Polk Run STP	First order product	0.946	73.1 +/- 3.5	0.53 +/- 0.08	NA	NA	1.30
Sycamore STP	First order logistic	0.994	66.7 +/- 1.8	0.50 +/- 0.07	NA	-0.57 +/- 0.08	1.38
Mineralisation
Polk Run STP	3/2 order	0.984	42.0 +/- 2.2	0.52 +/- 0.06	0.22 +/- 0.07	NA	NA
Sycamore STP	First order logistic	0.989	42.5 +/- 1.2	0.47 +/- 0.08	NA	-0.69 +/- 0.12	1.47

(a) A, initial parent concn (first-order decay), asymptote (first-order production and first-order logistic), or deflection point (3/2 order). k1, first-order rate constant. k0, zero-order rate constant. n, empirtical constant (unitless)

(b) Based upon first order rate constant

Data on the loss of parent (%) were fitted to various decay equations using nonlinear regression. The function, which statistically and visually provided the best fit for both sludges, was the simple first-order decay model, which has the form Y ) A exp-k1t, where Y equals % parent at time (t), A equals initial parent (%), and k1 equals first-order rate constant. In the case of mineralization, the Polk Run data were best described by a 3/2 order model without growth, which has the form Y ) A(1 - exp-k1t) + k0t, where Y equals % CO2 at time (t), A reflects the deflection point (%) between first- and zero-order kinetics, k1 equals the first-order rate constant, and k0 equals the zero-order rate constant. The mineralization data from Sycamore sludge were best described by a first- order logistic model, which has the form Y ) A(1 - exp-k1t)-1/n, where Y equals % CO2 at time (t), A equals the asymptotic yield of CO2 (%), k1 equals the first-order rate constant, and n equals a unitless empirical -constant. Inthecaseofcomplete degradation, which represents the percent mineralized or incorporated into biomass, the Polk Run data were best described by a simple first-order production, which has the form Y ) A(1 - exp-k1t), where Y equals % CO2 + % biomass (%) at time (t), A equals the asymptotic yield of CO2 + biomass, and k1 equals the first-order rate constant, while that from Sycamore sludge were best described by the first-order logistic model.

Validity criteria fulfilled:

not applicable

Interpretation of results:

readily biodegradable

Conclusions:

The following conclusions can be drawn from this study:
Within 8 h, 41-44% was evolved as 14CO2, 1-2% remained as parent, 24-33% was incorporated into biomass, and 10- 15% was present as intermediates, primarily sulfophenyl- carboxylates. Primary and complete biodegradation were best described by a first-order model. First-order rate constants for LAS were 0.96-1.10 h-1 for primary loss and 0.50-0.53 h-1 for complete degradation.
This study provides an accurate and comprehensive kinetic picture of biodegradation under realistic conditions as well as information on the mechanism of biodegradation.
The study demonstrates >60 % biodegradation (mineralisation and incorporation into biomass) is achieved within 52h when incubated in activated sludge under realistic WWTP conditions. Although this is not a classic ready biodegradability study, it demonstrates that the substance meets the ready biodegradability criteria.

Executive summary:

This study monitored the biodegradation of radiolabeled linear alkylbenzene sulfonate in activated sludge by specifically measuring parent, metabolites, and incorporation into biomass as a function of time. Within 8 h, 41-44% was evolved as 14CO2, 1-2% remained as parent, 24-33% was incorporated into biomass, and 10- 15% was present as intermediates, primarily sulfophenyl- carboxylates. Primary and complete biodegradation were best described by a first-order model. First-order rate constants for LAS were 0.96-1.10 h-1 for primary loss and 0.50-0.53 h-1 for complete degradation. Compared to a classic OECD 301 study, the data presented here provides less uncertainty regarding the interpretation of test results and an ability to assess the fate of chemicals, which are biotransformed but not mineralized.  Furthermore, the study provides an understanding of potential metabolites and kinetic data more relevant for exposure assessment. The study demonstrates >60 % biodegradation (mineralisation and incorporation into biomass) is achieved within 52h when incubated in activated sludge under realistic WWTP conditions.  Although this is not a classic ready biodegradability study, it demonstrates that the substance meets the ready biodegradability criteria.

Reference 4

Endpoint:

biodegradation in water: screening test, other

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

Reason / purpose for cross-reference:

read-across source

Parameter:

% degradation (test mat. analysis)

Value:

76

Sampling time:

52 h

Remarks on result:

other: Combined CO2 evolution and incorporation into biomass

Remarks:

Polk Run STP

Parameter:

% degradation (test mat. analysis)

Value:

66

Sampling time:

52 h

Remarks on result:

other: Combined CO2 evolution and incorporation into biomass

Remarks:

Sycamore STP

Validity criteria fulfilled:

not applicable

Interpretation of results:

readily biodegradable

Conclusions:

The following conclusions can be drawn from this study:
Within 8 h, 41-44% was evolved as 14CO2, 1-2% remained as parent, 24-33% was incorporated into biomass, and 10- 15% was present as intermediates, primarily sulfophenyl- carboxylates. Primary and complete biodegradation were best described by a first-order model. First-order rate constants for LAS were 0.96-1.10 h-1 for primary loss and 0.50-0.53 h-1 for complete degradation.
This study provides an accurate and comprehensive kinetic picture of biodegradation under realistic conditions as well as information on the mechanism of biodegradation.
The study demonstrates >60 % biodegradation (mineralisation and incorporation into biomass) is achieved within 52h when incubated in activated sludge under realistic WWTP conditions. Although this is not a classic ready biodegradability study, it demonstrates that the substance meets the ready biodegradability criteria.

Executive summary:

In a one-to-one read-across approach, the substance benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. (data on LAS as part of category approach) is considered appropriate for direct read-across (one-to-one) to benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts (target substance) for the endpoint biodegradation in water: screening test.

This study monitored the biodegradation of radiolabeled linear alkylbenzene sulfonate in activated sludge by specifically measuring parent, metabolites, and incorporation into biomass as a function of time. Within 8 h, 41-44% was evolved as 14CO2, 1-2% remained as parent, 24-33% was incorporated into biomass, and 10- 15% was present as intermediates, primarily sulfophenyl- carboxylates. Primary and complete biodegradation were best described by a first-order model. First-order rate constants for LAS were 0.96-1.10 h-1 for primary loss and 0.50-0.53 h-1 for complete degradation. Compared to a classic OECD 301 study, the data presented here provides less uncertainty regarding the interpretation of test results and an ability to assess the fate of chemicals, which are biotransformed but not mineralized.  Furthermore, the study provides an understanding of potential metabolites and kinetic data more relevant for exposure assessment. The study demonstrates >60 % biodegradation (mineralisation and incorporation into biomass) is achieved within 52h when incubated in activated sludge under realistic WWTP conditions.  Although this is not a classic ready biodegradability study, it demonstrates that the substance meets the ready biodegradability criteria.

A full justification for the read-across approach is presented in IUCLID Section 13.2.

Reference 5

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study was conducted equivalent to OECD 301F using pilot plant activated sludge. Although not GLP, the study was well reported and is considered suitable for chemical safety assessment purposes..

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Activated sludge sample was taken from a pilot-plant during and aerated for a period of 48h to remove as much as possible the remaining carbon sources and ammonia. The sludge, mineral media and stock solutions of the target compounds were mixed in the sapromate cells to give an initial sludge concentration of 30 mg of dry solids/L. The pilot-scale activated sludge plant consisted of a completely mixed aeration tank (490 l) and a secondary settler (280 l). The plant treated pre-settled municipal wastewater.

Duration of test (contact time):

28 d

Initial conc.:

25.1 mg/L

Based on:

test mat.

Remarks:

LAS-C12

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The sludge, mineral media and stock solutions of the target compounds were mixed in the sapromate cells to give an initial sludge concentration of 30 mg of dry solids/L and 80mg/L of LAS, 25.1mg/L of which consists of LAS-C12. A test with a blank inoculum was included as well as a test with 109 mg/L of the easily biodegradable potassium hydrogen phthalate (PHP) to serve as a method control. PHP is not one of the standard reference compounds in OECD test method 301F, but is mentioned in this method to represent compounds which require the addition of an inoculum to obtain biodegradation. The other reference compounds will also cause biodegradation, even when an inolculum is not deliberately added. All the tests were carried out in duplicate over a period of 28 days at a temperature of 20 C.

Reference substance:

other: potassium hydrogen phthalate (PHP)

Parameter:

% degradation (O2 consumption)

Value:

> 60

Sampling time:

28 d

Remarks on result:

other: 10-day window criteria met

Details on results:

The O2-consumption data generated in the ready biodegradability tests showed that LAS is readily biodegradable as defined by the OECD Guidelines: more than 60% of mineralisation was achieved within 28 days and this level was reached within a time window of 10 days from the start of mineralisation.

Results with reference substance:

Acceptable

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Conclusions:

As >60 % mineralisation of LAS-C12 was achieved wtihin 28-days, meeting the 10-day window criteria, the substance can be considered readily biodegradable.

Executive summary:

In a study conducted equivalent to OECD 301F, the ready biodegradability of linear alkylbenzene sulfonate (C12 -LAS) was assessed over a 28 -day period. As >60 % mineralisation of LAS-C12 was achieved wtihin 28-days, meeting the 10-day window criteria, the substance can be considered readily biodegradable.

Reference 6

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

Reason / purpose for cross-reference:

read-across source

Parameter:

% degradation (O2 consumption)

Value:

> 60

Sampling time:

28 d

Remarks on result:

other: 10-day window criteria met

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Conclusions:

As >60 % mineralisation of LAS-C12 was achieved wtihin 28-days, meeting the 10-day window criteria, the substance can be considered readily biodegradable.

Executive summary:

In a one-to-one read-across approach, the substance benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. (data on LAS as part of category approach) is considered appropriate for direct read-across (one-to-one) to benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts (target substance) for the endpoint biodegradation in water: screening test.

In a study conducted equivalent to OECD 301F, the ready biodegradability of linear alkylbenzene sulfonate (C12 -LAS) was assessed over a 28 -day period. As >60 % mineralisation of LAS-C12 was achieved wtihin 28-days, meeting the 10-day window criteria, the substance can be considered readily biodegradable.

A full justification for the read-across approach is presented in IUCLID Section 13.2.

Reference 7

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 June 2018 to 11 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with international guidelines and in a GLP laboratory. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Version / remarks:

Commission Regulation (EC) No. 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

Paragraph (m)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms was obtained on
11 June 2018 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Laboratory culture: No
- Method of cultivation: N/A
- Storage conditions: N/A
- Storage length: N/A
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection.
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 39.1 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured.
- Pretreatment: Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper (rinsed 3 times with 20 mL deionised reverse osmosis water prior to drying in an oven) using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC for at least 1-hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.3 g/L prior to use.
- Concentration of sludge: 30 mg suspended solids/L
- Initial cell/biomass concentration: N/A
- Water filtered: deionized reverse osmosis water used
- Type and size of filter used, if any: N/A

Duration of test (contact time):

28 d

Initial conc.:

8.4 mg/L

Based on:

act. ingr.

Remarks:

equivalent to 5 mg carbon/L

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: OECD Guideline-recommended composition used
- Additional substrate: No
- Solubilising agent (type and concentration if used): N/A
- Test temperature: 22 - 24 °C
- pH: 7.4 at test initiation
- pH adjusted: yes - using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all off the vessels being adjusted to 3 L by addition of mineral medium purged overnight with CO2-free air.
- CEC (meq/100 g): N/A
- Aeration of dilution water: yes
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes
- Other:

TEST SYSTEM
- Culturing apparatus: 5 litre culture vessels
- Number of culture flasks/concentration: inoculated control, procedure control (final concentration 10 mg carbon/L), test item (final concentration 5 mg carbon/L) - duplicate flasks; toxicity control (final concentration 15 mg carbon/L) - single vessel
- Method used to create aerobic conditions: The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/minute per vessel and stirred continuously by magnetic stirrer. The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
- Method used to create anaerobic conditions: N/A
- Measuring equipment: pH: Hach HQ40d Flexi handheld meter; samples were analyzed for IC using either a Shimadzu TOC-VCSH TOC analyzer or a Shimadzu TOC-LCSH TOC analyzer
- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water.
- Other:

SAMPLING
- Sampling frequency: Days 0, 7, 14, 21 and 28; (Inorganic carbon: Days 0, 2, 6, 8, 10, 14, 21, 28 and 29; pH: Day 0 and on Day 28 prior to acidification with hydrochloric acid)
- Sampling method: All samples were analyzed for Inorganic Carbon immediately. The remainder of all samples were frozen for further analysis if required. On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29. Samples for IC (50 or 135 μL) were injected into the IC channel of the TOC analyzer.
- Sterility check if applicable: N/A
- Sample storage before analysis: N/A
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: Not required
- Toxicity control: Yes (test item + reference). An aliquot (163 mL) of the test item stock solution was dispersed in inoculated mineral medium along with an aliquot (51.4 mL) of the sodium benzoate stock solution. The volume was adjusted to 3 liters to give a final concentration of 16.3 mg test item/L (equivalent to 8.4 mg ai/L) plus 17.1 mg sodium benzoate/L, equivalent to a total of 15 mg carbon/L.
- Other: Procedure control: reference item - sodium benzoate (C6H5COONa), was used. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium prior to the volume being adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.


STATISTICAL METHODS:
The theoretical amount of carbon present in each component was calculated as follows:
[(No. of C atoms * mol wt of C) / mol wt of test item] *100 mol wt of test item
The total percentage carbon content of the test item was 30.63 % (corrected for purity).
For a 10 mg C/L test concentration, which was equivalent to 16.3 mg test item/L (equivalent to 8.4 mg ai/L), the total organic carbon present for the test item was 15 mg C in 3 L.

The theoretical amount of carbon present in the reference item, sodium benzoate (C6H5COONa) was calculated as follows:
[(No. of C atoms * mol wt of C) / mol wt of sodium benzoate] *100
The theoretical amount of carbon present in the reference item was 58.34 %.
For a 10 mg C/L test concentration, which was equivalent to 17.1 mg reference item/L, the total organic carbon present for sodium benzoate was 30 mg C in 3 L.

The percentage biodegradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values into the following equation. The values of Replicates 1 and 2 are meaned for the inoculum control, test and reference items before substitution into the following equation:
% ThCO2 (= % biodegradation where conversion factor for C to CO2 is 3.67) = [(mg IC in test flask - mg IC in control flask) / mg TOC added as test chemical] * 100

The total CO2 evolution in the inoculum control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation:
Total CO2 evolution (mg C/L) = mg IC in control * (100 / %C of CO2) * (1 / test volume)

Reference substance:

benzoic acid, sodium salt

Preliminary study:

In order to investigate whether the test item adsorbed to filter matrices and/or the activated sewage sludge the following samples were analyzed for Organic Carbon using a Shimadzu TOC-VCPH TOC analyzer. The results indicated that the test item may adsorb to filter matrices but did not adsorb to activated sewage sludge. Therefore, for the purpose of the study, the samples taken for DOC analysis were centrifuged to remove the suspended solids present without causing a loss of any test item.

Test performance:

The test satisfied all the validation criteria as outlined in the 'methods and materials' fere text box.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

111

Sampling time:

28 d

Remarks on result:

other: Values in excess of 100% were considered to be due to sampling and/or analytical variation.

Details on results:

- The test item attained 111% biodegradation after 28 days and satisfied the 10-Day window validation criterion
- The toxicity control attained 73% biodegradation after 14 days and 83% biodegradation after 28 days.
- The total CO2 evolution in the inoculum control vessels on Day 28 was 34.82 mg/L.
- The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content.
- The difference between the values for CO2 production at the end of the test for the replicate vessels was <20%.
- The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of inoculum control Replicate 1 and test item Replicate 2.
- Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

Hence, all validity criteria as outlined in OECD Test Guideline 301B are satisfied.

Results with reference substance:

Sodium benzoate attained 88% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 96% biodegradation was attained. The validity criterion is satisfied.

Table 1.

Percentage biodegradation values

Day	Biodegradation %
	Procedure control	Test item	Toxicity control
0	0	0	0
2	55	21	41
6	73	55	60
8	84	78	75
10	85	91	87
14	88	97	73
21	71	87	73
28	85	114	75
29*	96	111	83

*Day 29 values corrected to include any carry-over of CO₂ detected in Absorber 2

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the relevant Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO₂ present in the test vessels. Therefore any additional CO₂ detected in the Day 29 samples originated from dissolved CO₂ that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.

Table 2. IC values on each analysis occasion

Day	Inorganic carbon (mg IC)
	Inoculum control				Procedure control				Test item				Toxicity control
	Replicate 1		Replicate 2		Replicate 1		Replicate 2		Replicate 1		Replicate 2		Replicate 1
	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	2.10	2.45	2.10	2.45	2.10	2.33	2.10	2.45	2.33	2.45	2.45	2.45	2.45	2.45
2	5.57	-	7.89	-	24.13	-	22.27	-	10.67	-	9.16	-	25.06	-
6	11.07	-	14.76	-	36.56	-	33.33	-	22.03	-	20.18	-	39.79	-
8	12.27	-	16.28	-	42.20	-	36.92	-	26.72	-	25.23	-	48.16	-
10	14.02	-	19.72	-	43.32	-	41.61	-	31.69	-	29.30	-	55.98	-
14	18.59	-	21.65	-	44.88	-	48.28	-	34.11	-	35.36	-	53.04	-
21	25.01	-	26.25	-	45.63	-	48.00	-	41.35	-	35.94	-	58.70	-
28	29.01	-	28.00	-	46.93/53.42*	-	48.72/54.54*	-	47.15	-	44.13	-	62.27	-
29	27.95	-	29.62	3.83	55.89	3.83	59.34	3.94	47.32	4.18	43.09	4.29	65.75	4.18

Abs = CO₂ vessel

- = No value determined

* - Results from re-analysis of sample due to original result deemed to be erroneous

Table 3. Total IC values in the culture vessels on Day 0

Test vessel	Total carbon* mg/L	Inorganic carbon* mg/L	IC content % of TC
Test item 5 mg C/L R1	5.16/5.06**	0.92/-0.18**	18/0**
Test item 5 mg C/L R2	5.26/4.68**	0.86/-0.30**	16/0**

R = Replicate

*Corrected for control values. Negative values are due to measured concentrations being less than control values

** Results from analysis of frozen sample due to erroneous results from original sample

Table 4. pH values

Test vessel	pH
	Day 0 (pre-adjustment)	Day 0 (post-adjustment)	Day 28
Inoculum control R1	7.7	7.4	7.5
Inoculum control R2	7.7	7.4	7.4
Procedure control R1	7.7	7.4	7.5
Procedure control R2	7.7	7.4	7.5
Test item control R1	7.7	7.4	7.4
Test item control R2	7.7	7.4	7.5
Toxicity control	7.7	7.4	7.5

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item attained 111% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%.

Executive summary:

A test was performed in accordance with OECD 301B (1992), whereby the test item was assessed for its biodegradability in an aerobic aqeous medium.

The test item, at a concentration of 5 mg carbon/L equivalent to 8.4 mg active ingredient (ai)/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels, in the dark, at temperatures of between 22 and 24 °C for 28 days. A reduced test concentration was employed as data supplied by the Sponsor indicated that the EC50 value for the test item for total respiration (corrected for the water content of the test item) was 169.2 mg/L with a No Observed Effect Concentration equal to or greater than 9.54 mg/L. The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

The toxicity control attained 73% biodegradation after 14 days and 83% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test. The reference attained 96 % biodegradation after 28 days, and satisfied the 10-d biodegradation window.

The test item attained 111% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B. Biodegradation values in excess of 100% were considered to be due to sampling and/or analytical variation.

Description of key information

Ready biodegradability (OECD 301B)

Ready Biodegradability, CO2 Evolution Test (Best, 2018) conducted on the registered substance benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts. The test item obtained 100 % biodegradation after 28 days and additionally satisfied the 10-day biodegradation window criteria. Based on these results, and given that the validity criteria were fulfilled, the test item can be considered readily biodegradable.

Further studies for this endpoint are presented in a weight-of-evidence utilising existing data on the read-across source substance benzenesulfonic acid, 4-C10-13-sec-alkyl derivatives, in support of the EU level conclusions drawn for EUSES modelling paramteres (HERA, 2013; SIDS, 2005).

Temmink and Klapwijk, 2004 conducted equivalent to OECD 301F, the ready biodegradability of linear alkylbenzene sulfonate (C12 -LAS) was assessed over a 28 -day period. As >60 % mineralisation of LAS-C12 was achieved wtihin 28-days, meeting the 10-day window criteria, the substance can be considered readily biodegradable.

Cavalli et al., 1993 presented the results of a 2 year monitoring programme assessing the removal and degradation of linear alkylbenzene sulphonate from a wastewater treatment plant are presented. The results refer to two surveys of the Torino WWTP , made in April 1989 and February 1990, that consisted of daily sampling programs. Inlets and outlets of the plant were totally sampled 12 times in April 1989 and eight times in February 1990. Samples were flow-proportional, average daily samplings taken hourly, automatically for waters and manually for sludges. Final dried sludges and sand were single samples taken occasionally. The specific measurements of LAS were completed with flow rate measurements of the various streams and with other unspecific measurements.

The main conclusions drawn from the LAS monitoring study on the WWTP of Torino can be summarised as follows:

Primary biodegradation in sewers is at least 12%.

Removal in the WWTP is 98 to 99%.

Primary biodegradation in the WWTP is in the range of 80 to 84 %,of which ultimate biodegradation is approximately 80%.

Federle et al., 1997 presented the biodegradation of radiolabeled linear alkylbenzene sulfonate in activated sludge was monitored by specifically measuring parent, metabolites, and incorporation into biomass as a function of time. Within 8 h, 41-44% was evolved as 14CO2, 1-2% remained as parent, 24-33% was incorporated into biomass, and 10- 15% was present as intermediates, primarily sulfophenyl- carboxylates. Primary and complete biodegradation were best described by a first-order model. First-order rate constants for LAS were 0.96-1.10 h-1 for primary loss and 0.50-0.53 h-1 for complete degradation. Compared to a classic OECD 301 study, the data presented here provides less uncertainty regarding the interpretation of test results and an ability to assess the fate of chemicals, which are biotransformed but not mineralized.  Furthermore, the study provides an understanding of potential metabolites and kinetic data more relevant for exposure assessment. The study demonstrates >60 % biodegradation (mineralisation and incorporation into biomass) is achieved within 52h when incubated in activated sludge under realistic WWTP conditions.  Although this is not a classic ready biodegradability study, it demonstrates that the substance meets the ready biodegradability criteria.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

As recognised in previous EU-level risk assessments for LAS-compounds in the category (including the registered substance benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts), EUSES modelling parameters can be modified based on substance specific information. The SimpleTreat model can consider the following distribution in a sewage treatment plant for the purpose of PEC estimation:

79% degradation

20% to sludge

1% release to water

HERA (2013) Human and Environmental Risk Assessment on Ingredients of Household Cleaning Products, Linear Alkylbenzene Sulphonate (CAS No. 68411 -30 -3), April 2013.

SIDS Initial Assessment Report for 20th SIAM, Linear Alkylbenzene Sulfonate (LAS), Paris, France, April 2005. UNEP Publications