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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
April 7 to May 5, 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study but with some details missing
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): a mixed population of sewage sludge treatment microorganisms from the secondary treatment stage of teh Severn Trent Water plc sewage treatment plant at Belper, Derbyshire, UK. Treating predominantly domestic sewage.
Duration of test (contact time):
28 d
Initial conc.:
6 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test media included: inoculated culture medium (control), 3 mg/L sodium benzoate with inoculum (positive control), 6 mg/L test material with inoculum (test), and 6 mg/L test material with 1.5 mg/L sodium benzoate with inoculum (toxicity control). Each of the 4 four media were inoculated with the activate sludge effluent at the rate of one drop of inoculum per liter (direct dispersal).

Standard culture medium was prepared by adding the following per liter of aerated reverse osmosis purified and deionized water that was left at room temperature (21C) and gently aerated for 24 hours prior to use:
11.76 g/L of CaCl2.2H2O, 4.93 g/L of MgSO4.7H2O, 2.59 g/L of NaHCO3, and 0.23 g/L KCl.
Reference substance:
benzoic acid, sodium salt
Test performance:
Positive Control, sodium benzoate biodegraded 98% after 28 days indicating the suitability of the test method and the culture conditions.
The Toxicity Control, sodium benzoate plus the test material biodegraded 70% after 28 days indicating the test material was not toxic to the sewage microorganisms.
The test material biodegraded 54% after 28 days.
Parameter:
% degradation (O2 consumption)
Value:
54
Sampling time:
28 d
Results with reference substance:
Positive Control, sodium benzoate biodegraded 98% after 28 days indicating the suitability of the test method and the culture conditions.
Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
The test substance is biodegradable but did not achieve the Guideline level of 60% in 28 days.
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1976
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Publication lacking some details; use of adapted sludge
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Qualifier:
equivalent or similar to
Guideline:
EU Method C.6 (Degradation: Chemical Oxygen Demand)
Deviations:
yes
Remarks:
use of adapted sludge; no replication
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
Innoculum from a sewage plant.
Preparation - daily 200 mL is separated fromthe 1L stock. After settling, the liquid is diluted with tap water, 600 mg/L starch or glucose, 600 mg/L peptone and 25 mL phosphate buffer (pH 7.2) plus the test compound.
The concentration of test compound was gradually increased to 200 mg/L as COD after 20 days
Mineral solution (medium) was 27.5 mg CaCl2, 22.5 mg MgSO4, 0.25 mg ferric chloride, 50 mg ammomium sulphate, 20 mL of phosphate buffer (pH 7.2) and 100 mL tap water in distilled water.
Duration of test (contact time):
>= 120 h
Initial conc.:
>= 0 - <= 200 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
not specified
Details on study design:
Test conducted in glass flasks.
One flask for test substance + inoculum + medium
One flask for analine + inoculum + medium
One flask for inoculum + medium
No aeration
Test temperature 20 +/- 3C
Reference substance:
aniline
Parameter:
other: COD
Value:
98.7
Sampling time:
120 h
Details on results:
rate of biodegradation equivalent to 8.4 mg COD/g/h
Results with reference substance:
94.5.% biodegradation. Rate equivalent to 19.0 mg COD/g/h
Interpretation of results:
inherently biodegradable
Conclusions:
p-toluene sulfonic acid is biodegradable to a substantial degree under the conditions of this test
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1976
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Publication lacking some details; use of adapted sludge; the concentration of the test substance (500 mg/L) and inoculum (5000 mg/L) are both higher than the OECD 302C guidelines, 30 mg/L and 100 mg/L, respectively. Only 72 h duration.
Principles of method if other than guideline:
Sewage treatment plant inoculum; adapted; 5000 mg/L inoculum in test; oxygen consumption as the parameter to measure biodegradation; 72 hour duration; 20C
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
Innoculum from a sewage plant.
Preparation - fill-and-draw unit containing 1.5L of mixed liquor; aerated to keep the floc in suspension. Every 24 hours the floc allowed to settle and 1L of supernatant wasted and replaced by 1L of nutrient solution. Nutrient solution was 500 mg/L dibasic potassium phosphate, 500 mg/L Calgonite, 325 mg/L ammonium phosphate and 50 mg/L ferric chloride in tapwater.
The concentration of test compound was gradually increased to 200 mg/L as COD after 20 days
Mineral solution (medium) was 27.5 mg CaCl2, 22.5 mg MgSO4, 0.25 mg ferric chloride, 50 mg ammomium sulphate, 20 mL of phosphate buffer (pH 7.2) and 100 mL tap water in distilled water.
Duration of test (contact time):
72 h
Initial conc.:
500 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
not specified
Details on study design:
Test conducted in glass Warburg flasks.
Test solution is 10 mL of adapted sludge suspension (5000 mg/L) and 10 mL of test substance solution (1000 mg/L in 2% phosphate buffer. Number of flasks per concentration was not indicated.
An inoculum only control was included in the study.
Test temperature 20C
ThOD was 1.287 mg/mg which is equivalent to 643.5 mg O2/L
Reference substance:
not specified
Details on results:
The oxygen uptake is lower for the test subtance than for the inoculum only control. The test substance is inhibiting the mircoorganisms.
Results with reference substance:
No data
Interpretation of results:
other: 500 mg/L of test substance inhibited microorganisms
Conclusions:
The test cannot be used to evaluate the biodegradability of the test substance as the concentration used, which is >10X the OECD guideline recommendation, inhibited the microorganisms.
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1976
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Publication lacking some details; use of adapted sludge
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Qualifier:
equivalent or similar to
Guideline:
EU Method C.6 (Degradation: Chemical Oxygen Demand)
Deviations:
yes
Remarks:
use of adapted sludge; no replicates
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
Innoculum from a sewage plant.
Preparation - daily 200 mL is separated fromthe 1L stock. After settling, the liquid is diluted with tap water, 600 mg/L starch or glucose, 600 mg/L peptone and 25 mL phosphate buffer (pH 7.2) plus the test compound.
The concentration of test compound was gradually increased to 200 mg/L as COD after 20 days
Mineral solution (medium) was 27.5 mg CaCl2, 22.5 mg MgSO4, 0.25 mg ferric chloride, 50 mg ammomium sulphate, 20 mL of phosphate buffer (pH 7.2) and 100 mL tap water in distilled water.
Duration of test (contact time):
>= 120 h
Initial conc.:
>= 0 - <= 200 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
not specified
Details on study design:
Test conducted in glass flasks.
One flask for test substance + inoculum + medium
One flask for analine + inoculum + medium
One flask for inoculum + medium
No aeration
Test temperature 20 +/- 3C
Reference substance:
aniline
Parameter:
other: COD
Value:
98.5
Sampling time:
120 h
Details on results:
rate of biodegradation equivalent to 10.6 mg COD/g/h
Results with reference substance:
94.% biodegradation. Rate equivalent to 19.0 mg COD/g/h
Interpretation of results:
inherently biodegradable
Conclusions:
Benzensulfonic acid is biodegradable to a substantial degree under the conditions of this test
Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1989
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Published result with minimal detail available
Qualifier:
no guideline available
Principles of method if other than guideline:
Aquifer microorganisms under dark, anaerobic conditions for 13 months. Measure for loss of test substance.
GLP compliance:
not specified
Oxygen conditions:
anaerobic
Inoculum or test system:
other: aquifer microorganisms
Details on inoculum:
Aquifer slurry from two sites near a municipal landfill: a methanogenic site (TOC 288 mg/L and sulfate concentration <0.1 mM) and a sulfate reducing site (TOC 14.4 mg/L and sulfate concentration 2.1 mM).
Duration of test (contact time):
13 mo
Initial conc.:
0.2 mmol/L
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
Experiments performed in the dark at room temperature in duplicate with sterilized aquifer slurries as controls.
Reference substance:
not specified
Details on results:
No degradation was observed.
Sulphate reducing slurry at 0 and 13 months was 205 uM and 198 uM, respectively
Methanogenic slurry at 0 and 13 months was 204 uM and 196 uM, respectively

No degradation was observed.

Sulphate reducing slurry at 0 and 13 months was 205 uM and 198 uM, respectively

Methanogenic slurry at 0 and 13 months was 204 uM and 196 uM, respectively

Conclusions:
No anaerobic degradation was observed under simulated aquifer conditions
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Secondary source - literature review with some details and references for numerous studies
Principles of method if other than guideline:
Six published studies; see results
GLP compliance:
not specified
Details on results:
A test of PTSA's biodegradability (100 mg/L) with non-adapted activated sludge (30 mg/L) as inoculum, at 20C +/- 1 and pH 7, biological oxygen demand was measured by respirometer over a period of 14 days (BOD14). Following a lag period of 50-60 hours and a degredation time of 125 to 135 hours, at a rate constant of 0.041 - 0.045 h-1, PTSA was found to be 79-80% biodegradable (Urano and Kato, 1986).

In a modified OECD screening test and in a coupled units test (both without further detail) the degree of sodium toluene sulphonate degradation was determined by measurements of DOC. The results were 99.9% and 88 +/- 3.14%, respectively (Schoberl and Huber, 1988). Accordingly, sodium toluene sulphonate is classified as being readily biodegradable.

In the OECD test for inherent biodegradability, according to test guideline 302B (Zahn-Wellens Test) at a concentration of 720 mg/L PTSA monohydrate was eliminated to 95% after 15 days (Wellens, 1979), while PTSA from the 65% aqueous solution at a concentration of 307.5 mg/L was eliminated to almost 100% after 5 days (Wellens, 1983).

In another study (Zahn-Wellens Test) PTSA monohydrate was degraded to 90% after 10 days and 94% after 20 days while the 65% solution was degraded to 75% after 25 days. When the test with the 65% aqueous solution was repeated with an adapted activated sludge, over 90% degradation was achieved after 10 days (Wellens, 1981).

In a recently published paper (1990) Wellens reports on the course of biodegradation of PTSA in the Zahn-Wellens test according to OECD Fuideline 302B and DIN-Norm 38412 (L12). The results showed 97% degradation after a maximum of 12 days incubation, whereby following an adaptation phase of 1-3 days, 85% degradation took place within 5-9 days.
Conclusions:
p-toluene solphonic acid is readily biodegradable
Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1980
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Published result with minimal detail available
Qualifier:
no guideline available
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
not specified
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
Two hundred sixty (260) of the existing chemicals listed by MITI have been tested for biodegradability.
Reference substance:
not specified
Details on results:
Test substance is reported to be biodegradable; although the presence of the sulfonic acid group was indicated (in structure activity correlations) was indicated to decrease (slow) the degradability of aromatic substances.
Conclusions:
Benzenesulphonic acid is degradable
Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1999
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Published result with minimal detail available
Qualifier:
no guideline followed
Principles of method if other than guideline:
DOC under anaerobic conditions at 37C for 8 weeks in a man-made sludge
GLP compliance:
not specified
Oxygen conditions:
anaerobic
Inoculum or test system:
other: aquifer microorganisms
Details on inoculum:
laboratory made sludge
Duration of test (contact time):
8 wk
Initial conc.:
100 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
Anaerobic at 37C; Test substance was incubated with 10 mL of laboratory-made sludge suspension (TOC 158.6 mg/L) for 8 weeks.
Reference substance:
not specified
Details on results:
No degradation was observed.
The gas volume produced was very similar to that of the blank. At 100 mg/L the test substance was slighty inhibitory (<=25%) to the microorganisms used.
Conclusions:
No anaerobic degradation was observed under the conditions of this laboratory-made sludge test.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
September 18 - October 16, 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP compliant; full documentation
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): activated sludge from aeration pool of local wastewater treatment plant
- Storage conditions: aerated in carboy until use
- Storage length: less than 30 days
- Preparation of inoculum for exposure: the sludge was maintained by the daily addition of synthetic sewage food; pH adjusted with dilute HCl to maintain a range of 6.5 - 8.0; dissolved oxygen maintained at least 2.0 mg/L;
- Pretreatment: preacclimation phase was 9 days in duration
- Concentration of sludge: 500 mL from each of two SCAS units combined
- Initial cell/biomass concentration:
- Water filtered: yes
- Type and size of filter used, if any: LABCONCO Water Pro Water Purifier PS
Duration of test (contact time):
>= 28 d
Initial conc.:
>= 10 - <= 20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: inorganic nutrient medium (see Table 1)
- Additional substrate: no
- Solubilising agent (type and concentration if used): no data
- Test temperature: 22+/- 3 degrees C
- pH: 7
- pH adjusted: no
- CEC (meq/100 g): no data
- Aeration of dilution water: stir bar
- Suspended solids concentration: used supernatant
- Continuous darkness: yes
- Other: acclimation in SCAS units; conducted acclimated and unacclimated tests in duplicate
Reference substance:
acetic acid, sodium salt
Preliminary study:
DOC analysis of the supernatant samples from the SCAS units indicated the inoculum had adapted to the test material within the 15-day period by the 82% degradation exhibited
Test performance:
49 mg CO2 evolved in the control system which is in the acceptable range per the guideline. Plate count analysis indicated the inoculum was viable and active.
Parameter:
% degradation (CO2 evolution)
Value:
>= 83 - <= 85
Sampling time:
28 d
Details on results:
The 60% threshold value was attained by the test substance by day 6 in both the 10 and 20 mg C/L test systems where the test substance SCAS acclimated inoculum was used. The carboys containing the nontest substance acclimated inoculum exceeded the 60% threshold by day 15 for both the 10 and 20 mg C/L units
Results with reference substance:
The 60% threshold value was attained by day 6. 93.2% of the theoretical CO2 evolution was attained at the end of the 28-day study.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
the test substance is readily biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
June 12 - July 11, 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP compliance; full documentation
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): activated sludge mixed liquors collected from aeration basin of local domestic wastewater treatment plant.
- Storage conditions: glass carboy and aerated
- Storage length: 19 hours
- Preparation of inoculum for exposure: a 1 liter sample of the 2.5 g/L sludge was homogenized using a blender. The sample settled for 30 minutes and supernatant was decanted and aerated until use (approximately 1 hour).
- Pretreatment: none
- Concentration of sludge: 2.5 g/L before homogenizing
- Initial cell/biomass concentration: 5.2 x 10-7 colony forming units / mL based on bacterial plate counts
- Water filtered: not specified
Duration of test (contact time):
>= 28 d
Initial conc.:
>= 10 - <= 20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: as detailed in Table 1
- Test temperature: 20-22 degrees C
- pH: no data
- Aeration of dilution water: aeration with CO2 free air
- Suspended solids concentration: no data
- Continuous darkness: yes
- Other:


TEST SYSTEM
- Culturing apparatus: 4 liter glass carboy
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aeration with CO2 free air
- Measuring equipment: Shimadzu Model 5050 total organic carbon analyzer with Shimadzu automatic sample injector Model 5000
- Test performed in closed vessels
- Details of trap for CO2 and volatile organics if used: 3 CO2 absorber gas-washing bottles in a series, each filled with 100 mL of 0.2 N KOH
- Other:


SAMPLING
- Sampling frequency: day 0,3,6,9,15,16,23,25 and 29
- Sampling method: KOH trap
- Sterility check if applicable: yes
- Sample storage before analysis: glass autosampler vials without headspace; caps were sealed with parafilm and stored at room temperature
- Other:


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: no data
- Other:


STATISTICAL METHODS: non-linear regression fit test
Reference substance:
other: Dextrose
Test performance:
Bacterial plate counts confirmed the presence of a significant viable bacterial population. The total mg CO2 evolved from the inoculum blank was within the limits specified in the protocol (i.e., <40mg CO2/L).
Parameter:
% degradation (TOC removal)
Value:
>= 69 - <= 87
Sampling time:
29 d
Details on results:
The four test replicates (two at 10 mg C/L and two at 20 mg C/l) yielded 29-day %TCO2 values of 84, 87, 82 and 69%, respectively. The 40-45% TCO2 was reached at 10 days. It took 16 days to reach 60% in all four replicates.
Results with reference substance:
113% TCO2 observed for the dextrose test systems
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
The test substance is readily biodegradable but required 16 days to reach 60% TCO2
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
September 18 - October 16, 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline test; GLP compliant; full documentation
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): aeration pond of local wastewater treatment plant
- Laboratory culture:
- Method of cultivation:
- Storage conditions: aerated in carboy in the laboratory
- Storage length: approximately 30 days
- Preparation of inoculum for exposure: the sludge was maintained by the daily addition of synthetic sewage food; pH adjusted with dilute HCl to maintain a range of 6.5 - 8.0; dissolved oxygen maintained at least 2.0 mg/L;
- Pretreatment: preacclimation phase was 9 days in duration
- Concentration of sludge: 500 mL from each of two SCAS units combined
- Initial cell/biomass concentration:
- Water filtered: yes
- Type and size of filter used, if any: ABC reagent water (equivalent to ASTM Type I) was used in the test. The water was purififed by a LABCONCO Water Pro Water Purifier PS.
Duration of test (contact time):
>= 28 d
Initial conc.:
>= 10 - <= 20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: inorganic nutrient medium (see Table 1)
- Additional substrate: no
- Solubilising agent (type and concentration if used): no data
- Test temperature: 22+/- 3 degrees C
- pH: 7
- pH adjusted: no
- Aeration of dilution water: stir bar
- Suspended solids concentration: used supernatant
- Continuous darkness: yes
- Other: acclimation in SCAS units


TEST SYSTEM
- Culturing apparatus: sodium acetate carboy
- Number of culture flasks/concentration: one
- Method used to create aerobic conditions: aerate with CO2 free air
- Test performed in closed vessels
- KOH traps used
- Other: Shimadzu Model 5050 Total Carbon Analyzer used for CO2 analysis (combustion at 680 degrees C and nondispersive infrared detection


SAMPLING
- Sampling frequency: days 2,4,6,8,10,15,20,25 and 28
- Sampling method: CO2 produced in the test systems was trapped in KOH solutions in the gas-washing bottles
- Sterility check if applicable: no data
- Sample storage before analysis: 7 mL aliquats in glass scintillation vials without headspace; teflon-lined cap; room temperature
- Other: standard plate count to enumerate the microbial populations was also performed


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: no data
- Other: reference material - sodium acetate


STATISTICAL METHODS: no data
Reference substance:
acetic acid, sodium salt
Preliminary study:
DOC analysis of the supernatant samples from the SCAS units indicated the inoculum had adapted to the test material within the 15-day period by the 81% degradation exhibited
Test performance:
49 mg CO2 evolved in the control system which is in the acceptable range per the guideline. Plate count analysis indicated the inoculum was viable and active.
Parameter:
% degradation (CO2 evolution)
Value:
>= 103 - <= 109
Sampling time:
28 d
Details on results:
The 60% of CO2 threshold was achieved within a 6 day test period.
Results with reference substance:
The day 0 total carbon analysis value was 15.03 mg C/L. The 60% threshold value was attainged in the reference system (sodium acetate) by Day 6 verifying the microbial inoculum was viable and active. 93% of the theoretical CO2 was attained at the end of the 28 days
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
the test material, sodium cumenesulfonate, is readily biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2004-08-18 to 2004-12-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
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 test


TEST 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: none


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 3 vessels (without test substance with inoculum)
- Abiotic sterile control: not performed
- Toxicity control: yes, 1 vessel
- Other: none


STATISTICAL METHODS:
Reference substance:
benzoic acid, sodium salt
Remarks:
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.
Parameter:
% degradation (CO2 evolution)
Value:
99.8
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:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test was repeated for consideration of the degradation extent data of the first test, because the test item showed a too high degradation extents > 100%.
In the repetition the test item showed again high degradation extents (> 100%), but the degradation extents of the single test vessels were more parallel and there were no outlier like in the first test.
In principle the result of the first test was confirmed.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study but not GLP
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
The inoculum was prepared from an activated sludge sampled in a predominantly urban sewage treatment plant. The supernatant of this activated sludge, free of non-settlable particles, had a microorganism concentration of 10x8 germs viable per milliliter at 22 degrees Celcius.
Duration of test (contact time):
>= 28 d
Initial conc.:
>= 10 - 20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: deionized water plus 0.25 g/L ferric chloride, 22.5 g/L magnesium sulfate, 27.5 g/L calcium chloride, 65 g/L phosphate buffer, and 40 g/L ammonium sulphate. Test medium composed of 4 ml ferric chloride solution, 1 ml each of magnesium sulphate, calcium chloride and ammonium sulphate solutions, and 2 ml phosphate buffer solution per litre of deionized water.
- Additional substrate: none
- Solubilising agent (type and concentration if used):
- Test temperature: 22 +/- 2 degrees C
- pH:
- pH adjusted: yes/no
- CEC (meq/100 g):
- Aeration of dilution water: stir bars
- Suspended solids concentration:
- Continuous darkness: yes
- Other:


TEST SYSTEM
- Culturing apparatus: 5 liter amber carboys containing 3 liters of test medium
- Number of culture flasks/concentration: one each per test concentration; two blanks
- Method used to create aerobic conditions: stir bars
- Method used to create anaerobic conditions:
- Measuring equipment: carbon dioxide determination device
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used: trapped as BaC03
- Other:


SAMPLING
- Sampling frequency: Days 1,4,5,7,8,11,12,13,15,18,20,22,26,27,28
- Sampling method:
- Sterility check if applicable:
- Sample storage before analysis:
- Other:


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes - 2 flasks
- Abiotic sterile control:
- Toxicity control: no
- Other:


STATISTICAL METHODS:
Parameter:
% degradation (CO2 evolution)
Value:
>= 86 - 88
Sampling time:
28 d
Results with reference substance:
biodegradation of Analine was 89% in 28 days
Interpretation of results:
readily biodegradable
Conclusions:
readily biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
7 April to 5 May 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study without detailed documentation
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
not specified
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): secondary treatment stage of the Severn Trent Water plc sewage treatment plant at Belper, Derbyshire, treating predominantly domestic sewage.
- Laboratory culture: inoculated culture medium
- Method of cultivation: direct dispersion in culture medium
- Storage conditions: room temperature
- Storage length:
- Preparation of inoculum for exposure:
- Pretreatment:
- Concentration of sludge:
- Initial cell/biomass concentration:
- Water filtered: yes/no
- Type and size of filter used, if any:
Duration of test (contact time):
>= 28
Initial conc.:
>= 7 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: standard culture medium
- Additional substrate: sodium benzoate as reference substance
- Solubilising agent (type and concentration if used):
- Test temperature:
- pH:
- pH adjusted: yes/no
- CEC (meq/100 g):
- Aeration of dilution water:
- Suspended solids concentration:
- Continuous darkness: yes/no
- Other:


TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions:
- Method used to create anaerobic conditions:
- Measuring equipment: not mentioned
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used:
- Other:


SAMPLING
- Sampling frequency: 3 day intervals
- Sampling method: % of ThOD
- Sterility check if applicable:
- Sample storage before analysis:
- Other:


CONTROL AND BLANK SYSTEM
- Inoculum blank: inoculated culture medium
- Abiotic sterile control:
- Toxicity control: 7 mg/L test material plus 1.5 mg/L sodium benzoate with inoculum
- Other: 3 mg/L sodium benzoate with inoculum


STATISTICAL METHODS:
Test performance:
The toxicity control attained 50% degradation after 28 days confirming the test material was not toxic to the sewage treatment micro-organisms used in the study.
Parameter:
% degradation (O2 consumption)
Value:
>= 40
Sampling time:
28 d
Results with reference substance:
Sodium benzoate attained 98% degradation after 28 days
Interpretation of results:
inherently biodegradable, not fulfilling specific criteria
Conclusions:
The test substance attained 40% degradation (and was still increasing) after 28 days and therefore is inherently biodegradable but does not meet the ready biodegradable criteria under the guideline.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
7 April to May 5 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study without detailed documentation
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
not specified
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): secondary treatment stage of the Severn Trent Water plc sewage treatment plant in Belper, Derbyshire, treating predominantly domestic sewage.
- Laboratory culture: inoculated culture medium
- Method of cultivation: direct dispersion in culture medium
- Storage conditions: room temperature
- Storage length:
- Preparation of inoculum for exposure:
- Pretreatment:
- Concentration of sludge:
- Initial cell/biomass concentration:
- Water filtered: yes/no
- Type and size of filter used, if any:
Duration of test (contact time):
>= 28 d
Initial conc.:
>= 7 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: standard culture medium
- Additional substrate: sodium benzoate as reference substance
- Solubilising agent (type and concentration if used):
- Test temperature:
- pH:
- pH adjusted: yes/no
- CEC (meq/100 g):
- Aeration of dilution water:
- Suspended solids concentration:
- Continuous darkness: yes/no
- Other:


TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration:
- Method used to create aerobic conditions:
- Method used to create anaerobic conditions:
- Measuring equipment:
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used:
- Other:


SAMPLING
- Sampling frequency:
- Sampling method:
- Sterility check if applicable:
- Sample storage before analysis:
- Other:


CONTROL AND BLANK SYSTEM
- Inoculum blank:
- Abiotic sterile control:
- Toxicity control:
- Other:


STATISTICAL METHODS:
TEST CONDITIONS
- Composition of medium:
- Additional substrate:
- Solubilising agent (type and concentration if used):
- Test temperature:
- pH:
- pH adjusted: yes/no
- CEC (meq/100 g):
- Aeration of dilution water:
- Suspended solids concentration:
- Continuous darkness: yes/no
- Other:


TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions:
- Method used to create anaerobic conditions:
- Measuring equipment: not mentioned
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used:
- Other:


SAMPLING
- Sampling frequency: 3 day intervals
- Sampling method: % of ThOD
- Sterility check if applicable:
- Sample storage before analysis:
- Other:


CONTROL AND BLANK SYSTEM
- Inoculum blank: inoculated culture medium
- Abiotic sterile control:
- Toxicity control: 7 mg/L test material plus 1.5 mg/L sodium benzoate with inoculum
- Other: 3 mg/L sodium benzoate with inoculum


STATISTICAL METHODS:
Test performance:
The toxicity control attained 62% degradation after 28 days confirming the test material was not toxic to the sewage treatment micro-organisms used in the study.
Parameter:
% degradation (O2 consumption)
Value:
>= 50
Sampling time:
28 d
Results with reference substance:
Sodium benzoate attained 98% degradation after 28 days
Interpretation of results:
inherently biodegradable, not fulfilling specific criteria
Conclusions:
The test substance attained 50% degradation after 28 days and therefore is inherently biodegradable but does not meet the ready biodegradable criteria under the guideline.

Description of key information

Based on all available information from aromatic sulphonic acids and the hydrotropes, the substance is considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Although limited well documented biodegradation studies are available for the aromatic sulphonic acids, and with no clear conclusion on ready biodegradability, the conclusion of ready biodegradability is strengthened by the number of supporting literature tests and the fact that seven biodegradation studies are performed with the closely related hydrotropes (the salts) for which it was concluded that these are readily biodegradable. As the cation has a limited affect on the biodegradation potential, and in principle the salt gets dissociated when in contact with water thus forming the acid, it is considered justified to conclude that these substances are readily biodegradable, taking into account all the available information. That information includes the 14 studies with the acid forms and the seven studies with the salt forms, which are all summarized below.

Aromatic Sulphonic Acids-

Amongst all the biodegradability studies for the aromatic sulphonic acids, the most reliable (Klimish 2) study is a 1995 OECD 301D (Ready Biodegradation - Closed Bottle Test) with CAS 98-11-3 (Huntsman, 1995a). The source of the inoculum was a secondary treatment sewage plant. Sodium benzoate, the reference substance, attained 98% degradation after 28 days. Based on DO measurements, the test substance attained 54% degradation after 28 days which cannot be considered as ready biodegradable under the guideline's 60% criteria. The toxicity control confirmed at 6 mg/L the test substance was not toxic to the microorganisms.

A test of PTSA's biodegradability (100 mg/L) with non-adapted activated sludge (30 mg/L) as inoculum, at 20C +/- 1 and pH 7, biological oxygen demand was measured by respirometer over a period of 14 days (BOD14). Following a lag period of 50-60 hours and a degredation time of 125 to 135 hours, at a rate constant of 0.041 - 0.045 h-1, PTSA was found to be 79-80% biodegradable (Urano and Kato, 1986).

In the OECD test for inherent biodegradability, according to test guideline 302B (Zahn-Wellens Test) at a concentration of 720 mg/L PTSA monohydrate was eliminated to 95% after 15 days (Wellens, 1979), while PTSA from the 65% aqueous solution at a concentration of 307.5 mg/L was eliminated to almost 100% after 5 days (Wellens, 1983).

In another study (Zahn-Wellens Test) PTSA monohydrate was degraded to 90% after 10 days and 94% after 20 days while the 65% solution was degraded to 75% after 25 days. When the test with the 65% aqueous solution was repeated with an adapted activated sludge, over 90% degradation was achieved after 10 days (Wellens, 1981).

In a recently published paper (1990) Wellens reports on the course of biodegradation of PTSA in the Zahn-Wellens test according to OECD Fuideline 302B and DIN-Norm 38412 (L12). The results showed 97% degradation after a maximum of 12 days incubation, whereby following an adaptation phase of 1-3 days, 85% degradation took place within 5-9 days.

Two studies (Pitter, 1976) demonstrate ultimate biodegradability for aromatic sulphonic acids and were conducted with methods similar to OECD guidelines for aerobic activated sludge tests with 28 day durations, reference substances and controls. Neither study is fully documented and hence received a Klimish 4. The author reported benzenesulfonic acid and p-toluene sulfonic acid are biodegradable and achieving 98% and 94% in 28 days, respectively.

Another 2 biodegradation studies are considered "weight of evidence". They have relatively little documentation. A 1980 review of MITI tested chemicals reports CAS 98-11-3 as biodegradable under aerobic conditions. A 1988 study of industrial, adapted activated sludge reports 90% TOC removal for CAS 104-15-4.

While not a microbial toxicity study per se, a 1966 study conducted with 500 mg/L hydroxybenzene sulfonic acid, reported inhibition of the microorganisms in the aerobic activated sludge test. However, this study is labeled as "disregarded study" as the 500 mg/L concentration was inhibitory to the microorganisms and there was no biodegradation. OECD guidelines recommend 30 mg/L maximum concentration.

In addition, two anaerobic biodegradability tests were performed with aromatic sulphonic acids. A 1989 anaerobic test of aquifer microorganisms showed no biodegradation of CAS 98-11-3 in 13 months. A 1999 anaerobic test using a "lab-made sludge", showed no degradation of 100 mg/L of CAS 98 -11 -3, at 37C for 8 weeks.

Hydrotropes

 There are four Klimisch 1 studies on the biodegradability of the hydrotropes and all 4 indicate ready biodegradability. The studies are conducted with sodium toluene sulphonate, sodium cumene sulphonate, sodium xylene sulphonate and calcium xylene sulphonate. There are an additional three Klimisch 2 studies. The study with sodium xylene sulphonate indicates ready biodegradability. The studies with sodium cumene sulphonate and sodium xylene sulphonate indicate inherent biodegradability.Based on all available information from these seven studies, the hydrotropes, and by read-across the aromatic sulphonic acids, are considered to be readily biodegradable.

 

The key hydrotrope studies are:

A 1992 OECD Guideline study - 301B Modified Sturm Test - based on CO2 evolution (Ruetgers Nease, 1992). The study was conducted according to GLP requirements and is fully documented. The test substance (sodium xylene sulphonate - CAS No 1300-72-7) achieved the 60% biodegradation threshold by Day 6 and reached 83% of theoretical CO2 in 28 days.

A 1993 OECD Guideline study - 301B Modified Sturm Test - based on CO2 evolution with sodium cumene sulphonate (CAS No 28348-53-0) (Ruetgers-Nease, 1993). The study was conducted according to GLP requirements and is fully documented. The test substance achieved the 60% biodegradation threshold by Day 6 and reached 103-109% of theoretical CO2 in 28 days.

A 1994 OECD Guideline Study - 301B Modified Sturm Test - based on CO2 evolution study with calcium xylene sulphonate (CAS No 28088-63-3) (Ruetgers-Nease, 1994). The study was conducted according to GLP requirements and is fully documented. The test substance reached 82-87% CO2 evolution in 28 days but took 16 days to reach the 60% "ready biodegradability" threshold.

A 2004 OECD Guideline Study - 301B Modified Sturm Test - based on CO2 evolution study with sodium toluene sulphonate CAS No 12068-03-0) (Brunswik-Titze, 2004).. The study was conducted according to GLP requirements and is fully documented. The test substance reached 100 -115% CO2 evolution in 28 days and reached the 60% "ready biodegradability" threshold in 4 days.