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Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-08-18 to 2004-12-23
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Justification for type of information:
Please see Category Approach
Reason / purpose for cross-reference:
read-across source
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: municipal wastewater treatment plant Breisgauer Bucht, Germany- Laboratory culture: no- Method of cultivation: not applicable- Storage conditions: not mentioned- Storage length: 2 days (test 1) resp. 1 day (test 2)- Preparation of inoculum for exposure: The activated sludge was washed twice by settling the sludge, decanting the supernatant and resuspending the sludge in aerated tap water.- Pretreatment: aerated with CO2-free air at a rate of 50-100 ml/min overnight- Concentration of sludge: Dry solid of the activated sludge was determined as 2.96 g/l (test 1) and 3.9 g/l (test 2) by weight measurements after 2 h drying at 105 °C (mean of triplicate measurements)- Initial cell/biomass concentration: 30 mg dry solid/L - Water filtered: not mentioned
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS- Composition of medium: according to the guideline- Additional substrate: none- Solubilising agent (type and concentration if used): not used- Test temperature: mean 21.0 - 22.1 (first test) and 20.0 - 22.0 °C (second test)- pH: not determined- Aeration of dilution water: not mentioned- Suspended solids concentration: 30 mg/L- Continuous darkness: no, diffuse light during testTEST SYSTEM- Culturing apparatus: Gas wash bottles (2000 ml volume) with lateral connecting pieces for butyl rubber septum were used as reactors. The liquid volume was fixed as 1500 ml each. Mixing was performed by a magnetic stirrer with 2 cm stir bars.- Number of culture flasks/concentration: three vessels per concentration- Method used to create aerobic conditions: aerated by passage of CO2-free air (2.7 - 5.5 bubbles/second) - Measuring equipment: Inorganic carbon measurement with total carbon analyzer TOC-5000A, Shimadzu- Details of trap for CO2: The CO2 produced in the reactors was absorbed in two 250 mL gas wash bottles in series, each filled with 200 mL 0.2 M NaOH.SAMPLING- Sampling frequency: days 4, 7, 11, 14, 21, 28 and 28 after acidification- Sampling method: Sampling was performed through the lateral connecting pieces through the butyl rubber septum using 5 ml PE syringes. - Sample storage before analysis: not mentioned- Other: noneCONTROL AND BLANK SYSTEM- Inoculum blank: yes, 3 vessels (without test substance with inoculum) - Abiotic sterile control: not performed- Toxicity control: yes, 1 vessel- Other: noneSTATISTICAL METHODS:
Reference substance:
benzoic acid, sodium salt
20 mg/L DOC
Preliminary study:
not performed
Test performance:
The CO2-free air production system consisted of an air compressor, two 1000 ml gas wash bottles filled with dry soda lime, followed by one bottle filled with 0.1 M NaOH (sodium hydroxide) and one gas wash bottle filled with demineralised water. The CO2-free air was passed on to an air distributewith two input and 22 output channels and through PE-tubes. Gas wash bottles (2000 ml volume) with lateral connecting pieces for butyl rubber septums were used as reactors. The liquid volume was fixed as 1.500 ml each. Mixing was performed by a magnetic stirrer with 2 cm stir bars. In both tests 7.1 mL of a stock solution (10 g/L) of the test item were added into the three test vessels and the vessel for toxicity control, corresponding to a TOC concentration of 20 mg/l TOC. From the reference compound 5.15 mL of a stock solution of 10 g/L were added to the reference vessels and the vessels for toxicity control in both tests. The CO2 produced in the reactors was absorbed in two 250 ml gas wash bottles in series, each filled with 200 ml 0.2 M NaOH. Sampling was performed through the lateral connecting pieces through the butyl rubber septum using 5 ml PE syringes. The amount of CO2 released from the reactors is calculated through IC-measurements in the CO2-absorber while considering the amount of CO2 removed for IC-measurement.
Key result
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
other: Result of second test
Details on results:
The degradation of the toxicity control was 94.0% within 14 days and 100.2% within 28 days (after acidification) in test 2, so there was no inhibition of the inoculum caused by the test item.
Results with reference substance:
The reference compound reached the pass level for ready biodegradablity (60% ThCO2) within 4 days (test 2).

Table #1: Ultimate biodegradation in % ThCO2 (first test)

 days biodegradation of test flasks biodegradation of reference flasks toxicity control 
   #1  #2  #3  mean  #1  #2  #3  mean  
 4  2.7  -1.2  36.0  12.5  54.5  32.4  67.2 51.4   33.3
 7  84.0  89.5  78.5 84.0   72.7  72.2  87.7  77.5  88.7
 11  104.2  103.0  96.4  101.2  84.4  83.8  98.0  88.7  100.2
 14  93.6  107.5  95.8  99.0  84.9  86.7  99.2  90.3  105.3
 21  108.0  103.8  106.5  106.1  91.1  93.2  97.2  93.8  109.7
 28  123.4  118.4  91.7  111.2  91.2  94.8  98.7  94.9  108.7
 28 (after acidification)  136.4  113.1  95.6  115.0  92.2  101.5  96.3  96.7  116.2

Table #2: Ultimate biodegradation in % ThCO2 (second test)

 days biodegradation of test flasks biodegradation of reference flasks toxicity control 
   #1  #2  #3  mean  #1  #2  #3  mean  
 4  45.4  34.0  58.0  45.8  71.6  66.2  65.1  67.6  38.6
 7  74.3  79.5  78.6  77.5  82.6  78.1  75.5  78.7  71.5
 11  87.9  91.4  95.0  91.4  84.5  86.6  78.3  83.1  87.2
 14  101.1  100.9  104.3  102.1  91.8  96.2  92.7  93.6  94.0
 21  101.1  112.6  106.9  106.9  101.2  102.4  92.5  98.7  98.2
 28  116.1  113.2  105.4  111.6  91.1  91.5  93.1  91.9  93.1
 28 (after acidification)  94.2  108.0  97.1  99.8  101.1  105.5  102.4  103.0  100.2
Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
The test was repeated for consideration of the degradation extent data of the first test, because the test item showed a high degradation rate of > 100%.In the repeated test, the test substance showed complete degradation with a mean value of 99.8%. The results of the repeated test confirmed those of the first study, although the rate of degradation in individual test vessels was more parallel and there were no outliers like in the first test.

Description of key information

There is a single biodegradation study for sodium toluene-4-sulphonate as well as six studies for three closely related substances that demonstrate the hydrotropes are readily biodegradable.   
There are 5 additional guideline biodegradation tests for closely related substances discussed below. Three of the four Modified Sturm tests concluded "readily biodegradable". The remaining Modified Sturm test reached 82-87% CO2 in 28 days but did not achieve 60% CO2 until day 16. A Closed Bottle test concluded the test substance was inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

To determine the biodegradation characteristics of sodium toluene-4-sulphonate the following important studies have been conducted on substances in the same hydrotopes category:

- Brunswick-Titze (2004), sodium toluene-4-sulphonate

- Albright and Wilson (1995), sodium cumene sulphonate

- Ruetgers-Nease (1993), sodium cumene sulphonate

- Ruetgers-Nease (1994), calcium xylene sulphonate

- Albright and Wilson (1995), sodium xylene sulphonate

- Bazzon (1993), sodium xylene sulphonate

- Ruetgers-Nease (1993), sodium xylene sulphonate

Sodium toluene-4-sulphonate is a member of the Hydrotropes Category, which comprises substances used to solubilise complex formulas in water. It is structurally related to the substances of the OECD SIDS Hydrotropes category, comprising three sub-groups: the methyl, dimethyl and methylethyl benzene sulphonates (or the toluene, xylene and cumene sulphonates). In addition, there are differences in counter ions (i.e. Na+, NH4+ and Ca++) in commercial hydrotropes. The chemical reactivity of the different counter ions is not expected to significantly influence the physical and chemical behaviour of the compounds with regard to environmental fate, ecotoxicity and mammalian toxicity. The presence of one or two methyl groups or a methylethyl group on the benzene ring is not expected to have a significant influence on chemical reactivity. As defined in the ‘Read-Across Justification Document’ section 13, data provided for this category are representative of potassium p-cumene sulphonate and suitable for assessment purposes. Experimental data for the Hydrotrope category has been evaluated and trends in the environmental fate of these hydrotopes were used to meet the requirements for the biodegradation endpoint.


Experimental studies on four members of the hydrotrope category of substances confirm ready biodegradability for sodium toluene-4-sulphonate. The key study (Brunswick-Titze, 2004) using sodium toluene sulphonate (CAS 657-84-1) in OECD 301B guideline test reported almost complete biodegradation by the end of the 28-day test of 99.8% with more than 60% degradation recorded after 7 days. Two OECD 301D (closed bottle test) guideline studies were undertaken using sodium cumene sulphonate and sodium xylene sulphonate (both by Albright and Wilson, 1995) with each reporting >=50% and >=40% at 28 days, respectively. The substances were biodegradable, although did not achieve ready biodegradability. In studies by Ruetgers-Nease (1993) OECD 301B (CO2 evolution test) guideline tests reported complete biodegradation for sodium cumene sulphonate and between 83 and 85% biodegradation using sodium xylene sulphonate after 28 days. Ruetgers-Nease (1994) and Bazzon (1993) both undertook OECD 301B guideline tests using calcium xylene sulphonate and sodium xylene sulphonate and reported more than 69 to 87% after 29 days and 86 to 88% after 28 days, respectively.


It is concluded that given the similarity between sodium toluene-4-sulphonate and the hydrotrope substances tested using the OECD biodegradation test guidelines, as well as similar physico-chemical properties and structure, it is fully expected that sodium toluene-4 -sulphonate is readily biodegradable. The key study, supported by a number of biodegradation studies using hydrotropes, confirms that the hydrotrope category substances are readily biodegradable.


In addition, the OECD SIDS Report (2006) for the hydrotropes category concluded that “studies across the hydrotropes category demonstrate rapid and complete biodegradation under aerobic conditions and the hydrotopes are considered to be readily biodegradable according to OECD criteria”. The OECD SIDS Report conclusion supports the experimental evidence provided.