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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
other: experimental study with main component of the test substance
Adequacy of study:
key study
Study period:
2008-11-04 to 2008-12-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study according to OECD Guideline 301 B.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Bacteria collected from activated sludge of the sewage treatment plant of Romanshorn, Switzerland
- Preparation of inoculum for exposure: The preparation was carried out according to the method described in the guideline.
- Concentration of sludge: a 30 mg solids/mL inoculum solution (24.5 g wet sludge with 50 mL mineral medium) was added to mineral medium directly after preparation.
Duration of test (contact time):
29 d
Initial conc.:
0.26 µg/L
Based on:
other: radiolabelled test material
Initial conc.:
0.27 µg/L
Based on:
other: non-radiolabelled test material (used in toxicity control)
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
radiochem. meas.
Remarks:
HPLC-RAM
Details on study design:
TEST CONDITIONS
2400 mL of the mineral solution and 3 mL inoculum were aerated for 24 hours in the test vessel. In the 2403 mL mineral solution, 110 mL of the radiolabelled test substance application stock solution (final concentration 0.26 µg/L) were added and homogenized. This solution was given to the test vessel, filled up to 3000 mL with mineral medium and immediately connected to the CO2 traps.
- Test temperature: 22 ± 2°C

STOCK SOLUTIONS
- Radiolabelled test item: A small amount (covering the tip of a spatula) was dissolved in 200 mL methanol. More test item had to be added as the first activity measured by liquid scintillation counting (LSC) was too low. LSC measurements determined a concentration of 19.8 µg/mL test substance in the stock solution. An application stock solution was prepared by transferring 50 mL of the stock solution into a 500 mL volumetric lfask. The methanol was evaporated and the test item resolved in 500 mL mineral medium. The final application stock solution was measured with LSC and was determined to have a concentration of 0.0072 µg/mL.
- Toxicity control: 79.59 mg of the non-radiolabelled test item were dissolved in 10 mL methanol. One mL of this first stock solution was diluted to 100 mL with methanol. To obtain the final application stock solution, 1.0 mL of the second stock solution was evaporated under a stream of nitrogen to complete dryness and resolved in 100 mL mineral medium. The toxicity control also contained the reference compound. Final concentration of the non-radiolabelled test item was 0.27 µg/L.
- Reference item: For the procedure control and the toxicity control two stock solutions were prepared on day 0 by dissolving 102.74 and 102.94 mg of sodium benzoate in 250 mL mineral medium, respectively. Final concentration was 20 mg TOC/L.
- Other:

TEST SYSTEM
- Culturing apparatus: 5 Liter flasks equipped with gas inlet and magnetic stirrer.
- Number of culture flasks/concentration: 2 for test suspension with radiolabelled test item, 2 for the inoculum blanks, 1 for procedure control and 1 for toxicity control
- Method used to create aerobic conditions: aerated with air free of carbon dioxide.
- Measuring equipment: carbon analyzer; HPLC-RAM was used for measuring the radioactivity present in the test suspension
- Details of trap for CO2 and volatile organics if used: Determination of the radioactivity from the KOH traps and determination of the absorbed CO2 in the Ba(OH)2 traps on the days 1, 2, 3, 6, 8, 10, 14, 17, 22, 28 and 29

SAMPLING
- Sampling frequency: 1, 2, 3, 6, 8, 10, 14, 17, 22, 28 and 29 days

CALCULATIONS:
- The biodegradation was calculated on the basis of the theoretical carbon content of the test substance and the cumulative quantities of carbon dioxide determined on the days of measurements. For the calculation the formula given in the guideline was used. For the blank values, the blank control with the vehicle was used.

Reference substance:
benzoic acid, sodium salt
Remarks:
20 mg TOC/L (procedure control)
Reference substance:
benzoic acid, sodium salt
Remarks:
20 mg TOC/L (toxicity control)
Key result
Parameter:
% degradation (CO2 evolution)
Value:
17.8 - 19.3
Sampling time:
29 d
Remarks on result:
other: Dose: 0.26 µg/L
Parameter:
% degradation (radiochem. meas.)
Value:
39.2 - 48.5
Sampling time:
29 d
Remarks on result:
other: Results are % of radioactivity present after 29 days referring to transfomation products of the radiolabelled test item
Details on results:
The biodegradation calculated as percentage of measured amount of carbondioxide over the theory was:
0.26 µg test substance/L = 17.8 - 19.3% in 29 days.

In addition, the transformation of the radiolabelled test item into its transformation and degradation product was measured as radioactivity via HPLC-RAM. After 29 days only 51.5% and 60.8% of the radioactivity present consisted of the radiolabelled test item. The remaining radioactivity, i.e. 48.5% and 39.2%, consisted of metabolites. From the HPLC-RAM chromatogram of the two test suspensions the detected metabolites are considered to be phenole (at a retention time of 2.6 minutes both), phenylthiophosphate (at a retention time of 10.3 and 12.8 minutes) and phenylphosphates (at a retention time of 14.5 and 14.8 minutes).
Results with reference substance:
procedure control = 90.6% in 14 days.
toxicity control = 98.1% in 29 days (87.7% in 14 days)
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
other: experimental study with main component of the test substance
Adequacy of study:
key study
Study period:
2008-09-02 to 2008-10-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Deviations:
yes
Remarks:
, radiolabelled substance was used. Aeration was not done with purified and humidified air. Losses due to evaporation were replaced by filling up with deionized water.
GLP compliance:
yes (incl. QA statement)
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): obtained from the sewage treatment plant Romanshorn, Switzerland
- Preparation of inoculum for exposure: centrifuged at 7380 rpm and 20°C for 5 minutes. Supernatant was discarded and the procedure repeated until sufficient sludge was concentrated. The sludge was washed twice with mineral medium and the supernatant discarded. The moisture content was determined by drying.
- Concentration of sludge: To obtain a final concentration of 0.2 g solid/L in the test systems, approximately 3.65 g of the activated sludge were added to the test vessels
- Water filtered: no
Duration of test (contact time):
28 d
Initial conc.:
0.26 µg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
Remarks:
LSC analysis of applied radioactivity
Details on study design:
TEST CONDITIONS
- Composition of medium: as described in DOC Die-away, CO2 evolution, Manometric Respirometry and Modified OECD Screening methods of Guideline 301 (adopted 1992) for determining ready biodegrability
- Test temperature: 20 - 25°C
- Aeration of dilution water: yes
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Glass beakers with volume of 2 L, equipped with magnetic stirrer and glass tube to introduce purified air.
- Number of culture flasks/concentration: 2 for control and test substance, 1 for reference substance
For the test, a super stock solution was prepared by adding a small amount (covering a small spatula) of the test item to 200 mL methanol. The super stock solution was analyzed by liquid scintillation counting (LSC) to determine the total radioactive residue (TRR). To obtain a final test concentration of 0.24 to 0.28 µg/L, corresponding to 85000 an 10000 dpm/L, more test item had to be added to the stock solution. This procedure was repeated until the appropriate concentration was obtained. The final super stock concentration was 21.5 µg/200 mL.
To prepare the application stock solution, 15 mL of the super stock solution was transferred to a 500 mL volumetric flask and evaporated to complete dryness under a stream of nitrogen. The flask was filled with mineral medium to the mark. The solution was submitted to ultrasonic treatment for approximately 10 minutes before it was mixed for 30 minutes at 60°C. The total radioactivity was determined during the mixing process. The application stock solution had a final concentration of 0.0030 µg/mL. Prior to application the stock solution was mixed over night at room temperature.

SAMPLING
- Sampling frequency: after 3,5 hours, 2, 8, 12, 16, 23, 27 and 28 days
- Sampling method: 6 mL of samples were used for determination of degradation. Samples of the blank controls were filtered by special membrane filters, discarding the first mL. The resulting 5 mL were used for DOC determination. One drop of H3PO4 was added to each sample before they were stored in the freezer until use. The samples containing the test item were not filtered as the samplings of day 0 showed that the radioactivity was bound to the filter. Liquid-Scintillation-Counting (LSC) measurements were performed with the resulting 5 mL after filtration. Additionally, LSC was measured from samples without filtration. The results showed that the radioactivity was bound to the filter. Therefore, no filtration was performed from samples of the test suspensions at the remaining sampling intervals. As the test item concentration was too low for DOC measurements, no DOC was determined.
Since the radioactivity in the application solution was too low for HPLC/RAM analysis, only LSC measurements were performed.


Reference substance:
ethylene glycol
Remarks:
165 mg TOC/L
Key result
Parameter:
% degradation (radiochem. meas.)
Value:
>= 59.5 - <= 66.8
Sampling time:
28 d
Remarks on result:
other: Dose: 0.26 µg/L
Details on results:
After 28 days, based on LSC analysis, recoveries of 37.1 and 29.9% of the initial applied concentration were found
Results with reference substance:
degradation based on measured DOC: 98.2% in 28 days
Validity criteria fulfilled:
yes
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented, according to accepted guideline, with GLP. Application of adsorbed test substance via glass filter paper. This test method is of limited applicability for not readily degradable and adsorptive substances.
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle 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 (e.g. location, sampling depth, contamination history, procedure): The aeration stage of the Anglian Water sewage treatment plant (Godmanchester) treating predominantly domestic sewage.
- Storage conditions: filtrate was maintained under aeration.
- Storage length: 24 h.
- Preparation of inoculum for exposure: sample was allowed to settle for approximately 30 minutes and the supernatant filtered through Whatman GFA coarse filter paper (first 250 ml discarded).
- Water filtered: yes
- Type and size of filter used, if any: aerated reverse osmosis purified water
Duration of test (contact time):
28 d
Initial conc.:
3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Standard nutrient medium
- Test temperature: 20°C
- Aeration of dilution water: aerated
- Other: test substance was dissolved in chloroform to give a stock solution of 840 mg/10 ml. Aliquots of stock solution were placed on individual pieces of Whatman GFA glass filter paper and the solvent allowed to evaporate to dryness. One piece of paper was placed in each test bottle prior to filling with inoculated medium.


TEST SYSTEM
- Culturing apparatus: (BOD) bottles (280 ml) of darkened glass and fitted with ground glass stoppers.
- Number of culture flasks/concentration: 18 bottles.
- Method used to create aerobic conditions: bottles were filled by siphon and firmly stoppered to exclude all air bubbles.
- Measuring equipment: Yellow Springs BOD Meter (Model 59); (COD) was determined using a semi-micro sample digestion method (Hach®).
- Test performed in open system: No


SAMPLING
- Sampling frequency: 0, 4, 7, 11, 14, 18, 21, 25 and 28 days
- Sampling method: BOD: duplicate bottles were removed for measurement of dissolved oxygen concentration.


CONTROL AND BLANK SYSTEM
- Inoculum blank: Inoculated nutrient medium and Inoculated nutrient medium plus filter paper.
- Toxicity control: 3 mg/l test substance plus filter paper and 3 mg/l sodium benzoate.
Reference substance:
other: Sodium benzoate
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
Points of degradation plot (test substance):
0 % degradation after 4 d
1 % degradation after 7 d
1 % degradation after 11 d
4 % degradation after 14 d
1 % degradation after 18 d
3 % degradation after 21 d
0 % degradation after 25 d
0 % degradation after 28 d
Parameter:
COD
Value:
1.39 other: mg O2/mg test mat.
Results with reference substance:
Points of degradation plot (reference substance):
55 % degradation after 4 d
64 % degradation after 7 d
58 % degradation after 11 d
63 % degradation after 14 d
61 % degradation after 18 d
68 % degradation after 21 d
66 % degradation after 25 d
58 % degradation after 28 d

The substance exhibited no signs of degradation during the test period.

Interpretation of results:
under test conditions no biodegradation observed
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1988-09-22 to 1988-11-01
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP guideline study. Deviations to the updated guideline (adopted 1992): reduced test volume solution, use of an emulsifier
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
(see principles of method other than guideline field)
Principles of method if other than guideline:
The volume of the test solution was reduced from 3.0 L to 1.5 L. The CO2 formed by biodegradation was absorbed with NaOH and determined on a carbon analyzer. Due to the poor solubility of the test substance in water, an emulsifier was used to achieve a better distribution in the medium. The test substance was added to the medium, homogenized with Nonylphenol 10E05P0. The CO2 production of the blank was 29.3 mg/L medium.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum / activated sludge: Bacteria collected from activated sludge of the sewage treatment plant of CH-4135 Reinach.
- Preparation of inoculum for exposure: The preparation was carried out according to the method described in the guideline.
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
test mat.
Initial conc.:
20 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
1200 mL of the mineral solution with the inoculum were aerated for 24 hours in the test vessels. In 300 mL mineral solution 0.5 mL Nonylphenol 10E05P0 (solution of 31.2 mg in 100 mL bidest. water) and 15 resp. 30 mg of test substance were added and homogenized. This solution was given to the test vessel which was immediately connected to the CO2 traps.
- Solubilising agent (type and concentration if used): Nonylphenol 10E05P0
- Test temperature: 22 ± 2°C

TEST SYSTEM
- Culturing apparatus: 2 Liter flasks equipped with gas inlet and magnetic stirrer
- Number of culture flasks/concentration: 1
- Method used to create aerobic conditions: approx. 25 mL/min free of carbon dioxide
- Measuring equipment: carbon analyzer
- Details of trap for CO2 and volatile organics if used: Determination of the initial CO2 of the 0.05 N sodium hydroxide and the CO2 absorbed in the absorbers filled with 200 mL 0.05 N sodium hydroxide on the days 5, 11, 18, 22, 27 and 28.

SAMPLING
- Sampling frequency: 5, 11, 18, 22, 27 and 28 days

CALCULATIONS:
-The biodegradation was calculated on the basis of the theoretical carbon content of the test substance and the cumulative quantities of carbon dioxide determined on the days of measurements. For the calculation the formula given in the guideline was used. For the blank values, the blank control with the vehicle was used.

ThOC:
- 10 mg test substance are equivalent to 6.315 mg organic carbon. This calculation is based on the technical formula: C18H15SPO3
Reference substance:
aniline
Remarks:
20 mg/L
Parameter:
% degradation (CO2 evolution)
Value:
2
Sampling time:
28 d
Remarks on result:
other: Dose: 10 mg/L
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Remarks on result:
other: Dose: 20 mg/L
Details on results:
The biodegradation calculated as percentage of measured amount of CO2 over the theory was:
10 mg test substance/L = 2% in 28 days
20 mg test substance/L = 0% in 28 days
Results with reference substance:
20 mg/L = 94.4% in 28 days
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, short documentation, high test item concentration
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
other: Inoculum according to MITI requirements
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Sludge collected from 10 different places in Japan
- Laboratory culture: cultivated for a month
- Concentration of sludge: 30 mg/L
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
test mat. analysis
Remarks:
HPLC
Parameter:
% degradation (O2 consumption)
Value:
-6
Sampling time:
28 d
Remarks on result:
other: mean out of 3 measurements
Parameter:
% degradation (test mat. analysis)
Value:
0
Sampling time:
28 d
Interpretation of results:
under test conditions no biodegradation observed

Description of key information

In conclusion, tests on the entire substance (EC 421 -820 -9) did not reveal a relevant biodegradation of the test substance. The very low solubility of the substance (or its single components) may have reduced the availability and hence the degradability of the substance in the test. However, for one of the main components (Structure A, CAS 597-82 -9) additional experimental data is available which revealed that this component is primarily degradable under formation of readily degradable degradation products.

Key value for chemical safety assessment

Additional information

The substance consists of several components:ForO, O, O-triphenyl phosphorothioate (CAS 597-82-0) additional tests on biodegradation are available. The fact that this compound is one of the main components of the present multi-constituent substance scientifically justifies the use of these studies for the assessment of the multi-constituent compound itself.

Five studies on the biodegradation in water are available. Four studies investigated the ready biodegradability and one investigated the inherent biodegradation.

 

Whole substance

Two supporting studies on ready biodegradability with the uvcb substance are available. In a Closed Bottle Test according to OECD 301D no degradation was observed after 28 days using 3 mg/L test substance (Huntington 1998).

In a study according to OECD 301C (Institute of Ecotoxicology, Gakushuin University 1999) no degradation was observed after 28 days using 100mg/L test item. Considering that the substance consists of several adsorptive constituents with poor water solubility, the limited bioavailability in the test design could be the reason for the lack of observable biodegradation.

In these studies the biodegradation of the single constituents was not analysed separately. With respect to the limited solubility of the main constituents, the bioavailability of the test item was limited and the applied methods not sensitive enough to measure degradation.

 

Structure A

In the key study onready biodegradability,the major constituent Structure A (CAS 597-82-0) was assessed over a 28 day period by the modified Sturm test (OECD 301B) (Springborn Smithers 2009). The nominal test concentration was 0.26 µg a.s./L (two replicates), which was in the range of the water solubility of the test substance. CO2measurements showed up to 17.8 and 19.3% mineralisation after 29 days. Further investigations during experimental phase using radiolabelled test item showed that after 29 days only 51.5% and 60.8% of the radioactivity present consisted of the test item, whereas the remaining radioactivity consisted of its degradation products. Thus, the test item is partially completely biodegraded over a 28 day period under the test conditions, up to 19% is mineralized and additionally up to 48.5% of the test item is transformed into its transformation and degradation products. The positive control substance of the ready biodegradation test (sodium benzoate) was 90.6 % degraded after 29 days. Phenol can be identified from the chromatogram of the degradation products: From the analytical measurements in this study it can be concluded that Phenol will be formed. RP HPLC was used and separation is based on hydrophobicity. The lower the content of phenolic groups of the analyte, the more polar the analyte is and eluation from the column starts earlier. Using 60% methanol at the beginning Phenol would correspond to the peak at 2.6 minutes. The peaks at 12.3 minutes correspond to the substance containing only two phenolic groups. (for further information see the attachments of the key study in IUCLID chapter 5.2.1, modified Sturm test, Springborn Smithers 2009).

This is supported by the results of the hydrolysis study with Structure A (see IUCLID section 5.1.2 , Ciba 2007) which showed that different transformation products of the test substances are generated in parallel once hydrolysis has started. Phenol was identified as degradation product.

The results indicate that the test item cannot be classified readily biodegradable but primarily degradable under conditions of the test.

 

Theinherent biodegradabilityof the radiolabelled test item was assessed over a 28 day period by the MITI-test (II) according to OECD 302C (Springborn Smithers 2008). The nominal test concentration was 0.26 µg a.s./L, which was in the range of the water solubility of the test substance. The test material attained up to 59.5 and 66.8 % biodegradation after 28 days whereas the positive control substance (ethylene glycol) was 98.2 % degraded 28 days. These results indicate that the test item can be classified as inherently biodegradable under conditions of the test.

 

In a supporting study, the ready biodegradability of Structure A was assessed over a 28 day period by the modified Sturm test (OECD 301B) in concentrations of 10 and 20 mg/L. No biodegradation (0 -2%) was observed. In line with the key study on ready biodegradation described above, the results indicate that the test item cannot be classified readily biodegradable under conditions of the test. However,the very low solubility of the substance may have reduced the availability and hence the degradability of the substance in the test. For characterisation of the persistency of the substance the test-set-up of the supporting study was not sufficient due to the low solubility of the test item.

 

Overall, the results of studies with the uvcb substance are not considered to be contradictory to the results of the key studies with the major constituent Structure A. The methodology of the studies performed with the uvcb substance was not sensitive enough to measure the degradability of the individual constituents.

The major constituent Structure A is primarily degradable under the conditions of the test. The determined degradation product phenol is readily biodegradable.