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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Test design not equivalent to current standard methods. However, it provides some scientific valid information to be taken into account for the weight of evidence analysis.
Principles of method if other than guideline:
The amounts of 14CH3- or 14C-phenyl-BHT (as an ethanol solution to give a well-suspended BHT) and activated sludge were added to the standard culture solution (100 mL) and each of the mixture was incubated aerobically by supplying CO2 free air continuously at the rate of 5 mL/min over 5-16 weeks at 25 ± 1 ºC in the dark. The 14CO2 trap was replaced weekly.
GLP compliance:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): 14CH3-BHT
- Radiochemical purity (if radiolabelling): > 99%
- Specific activity (if radiolabelling): 2.97 mCi/mmol

- Name of test material (as cited in study report): 14C-phenyl-BHT
- Radiochemical purity (if radiolabelling): > 99%
- Specific activity (if radiolabelling): 3.03 mCi/mmol
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: The sludge was obtained from the Chemical Inspection and Testing Association (Tokyo).
- Laboratory culture: Cultivated aerobically at 25 ± 2 ºC prior to use with feeding a synthetic sewage water (0.1% of glucose, peptone, and KH2PO4) according to the MITI´s procedure.

The activated sludge was included at different concentrations: 1, 3, 10, 30 and 100 ppm
Duration of test (contact time):
>= 5 - <= 16 wk
Initial conc.:
0.3 other: ppm
Initial conc.:
1 other: ppm
Initial conc.:
3 other: ppm
Initial conc.:
10 other: ppm
Initial conc.:
30 other: ppm
Parameter followed for biodegradation estimation:
CO2 evolution
Remarks:
14CO2
Details on study design:
The amounts of 14CH3- or 14C-phenyl-BHT (as an ethanol solution to give a well-suspended BHT) and activated sludge were added to the standard culture solution (100 mL) and each of the mixture was incubated aerobically by supplying CO2 free air continuously at the rate of 5 ml/min over 5-16 weeks at 25 ± 1 ºC in the dark. The 14CO2 trap was replaced weekly.

TEST CONDITIONS
- Composition of medium:
Aqueous solution containing:
65.3 ppm K2HPO4
25.5 ppm KH2PO4
53.1 ppm Na2HPO4
5.1 ppm NH4Cl
33.0 ppm MgSO4
85.5 ppm CaCl2
0.45 ppm FeCl3
- Additional substrate: None.
- Solubilising agent (type and concentration if used): Ethanol 0.2 mL/L.
- Test temperature: 25 ± 1ºC
- pH: No data.
- Aeration of dilution water: During test, mixtures were aerobically incubated by supplying CO2-free air continously at the rate of 5mL/min.
- Suspended solids concentration: The activated sludge was added at different concentrations: 1,3,10, 30 and 100 ppm.
- Continuous darkness: yes
- Other: The 14CO2 trap was replaced weekly.

TEST SYSTEM
- Culturing apparatus: Serially connected flasks (air-in), the first one was the CO2 absorber (20% NaOH, 2L), the second was distilled water (1L), the third one was distilled water (empty), the fourth was the incubation flask (200 mL) which included a stirrer and bath, the fifth was the polyurethane trap (c.a. 7g) and the last one was the 14CO2 trap (1N NaOH; 250 mL).
- Number of culture flasks/concentration: One apparatus (above-mentioned six flasks) per concentration.
- Method used to create aerobic conditions: CO2-free air was supplied at a continuous rate of 5mL/min.
- Measuring equipment: Liquid scintillation spectrometer (Packard, 3385).
- Test performed in closed vessels due to significant volatility of test substance: It was performed in closed vessels in order to assess the volatility.
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: Polyurethane trap ( c.a. 7g) and 14CO2 trap (250 mL of 1N NaOH).

SAMPLING
- Sampling frequency: 14CH3-BHT= 35 days. 14C-phenyl-BHT= 56-112 days.
- Sampling method:
- Sample storage before analysis: No data.
- Other: The polyurethane and 14CO2 traps were pretreated prior to radioactivity measurement and the incubation mixtures were centrifugated at 8000 rpm for 5min.

The activated sludge was extracted by adding 1N HCL/MeOH (1/3, v)
(30mL x3; 50mL x1 at reflux), the residue was exposed to an oxidizer after which the liquid scintillation counting (LSC) was conducted. 30 mL of saturated NaCL plus AcOEt (100 mL x3) was added to the HCl/MeOH fraction and the extract was obtained. The aqueous fraction was subjected to LSC and the AcOEt fraction was subjected to TLC and thereafter to LSC.

The supernatant was extracted by adding 5mL 1N HCl and 100 mL x3 AcOEt. The aqueous fraction was subjected to LSC and the AcOEt to TLC and then to LSC.

CONTROL AND BLANK SYSTEM
- Other: The publication does not report any kind of control.

Key result
Parameter:
% degradation (radiochem. meas.)
Value:
4.7
Sampling time:
28 d
Remarks on result:
other: Not ready biodegradable
Remarks:
23.1 % BHT volatilisation

The ratios of BHT/activated sludge (defined as BHT load) influenced the degree of biodegradation. As BHT load was lowered, residual BHT decreased and the evolution of 14CO2 increased proportionally. When BHT load was constant, lower concentrations of activated sludge made the biodegradation slower. This means that efficiency of contact between the chemical and microorganisms decreased as the concentrations of BHT and the sludge diminished.

From the results and from the facts that the absolute amount of 14CO2 evolved from each incubation mixture was proportional to the quantity of the added 14C-BHT, it is suggested that BHT was biodegraded both from solution and suspension and that rapidity of biodegradation was in the order of dissolved > well-suspended >> non-suspended.

Evolution of 14CO2 from 14CH3-BHT was greater than that from 14C-phenyl-BHT. This means that CH3 group was degraded more easily than benzene ring in molecule.

BHT volatilised from incubation flask and amounted to 20-28% (28-39% without ethanol) over 35 days, 27-33% over 56 days, and 45-51% over 112 days. Volatilization must be considered in calculating biodegradation.

The five major metabolites, formed at maximum amounts of 40% of the applied BHT, were identified as BHT-OOH, BHT-OH, BHT-CH2OH, BHT-CHO and BHT-COOH.

Validity criteria fulfilled:
not applicable
Interpretation of results:
not readily biodegradable
Conclusions:
According to the OECD Guidance on Aquatic Toxicity Testing of Difficult Substances and Mixtures (June 2000), the concentration of the solvent in the final test medium should not exceed 0,1 mL/L. However, in this experimental work and considering the very low solubility of BHT, a series of tests were carried out with radiolabeled BHT in different concentrations together with different concentrations of activated sludge and with 0.2 mL/L ethanol in an aerated system. Two experiments were carried out adding BHT without ethanol but only one was conducted using a concentration of BHT near the water solubility limit (1 mg/L). Therefore, only the results from this experiment should be taken into account. Slow primary degradation (5.2 % 14CO2 of 14C-CH3-BHT), residual BHT in solution (10.8 %) and volatilisation of BHT (26.2 %) after 35 days of incubation is reported. In order to classify a substance a readily biodegradable, taking into account the criteria established in the CLP Regulation, in a 28-day ready biodegradation study based on carbon dioxide generation, at least a level of 60 % of theoretical maximum.degradation must be achieved. The results provided in this experimental work refer to a period of 35 days. Therefore, it is difficult to assess the readily biodegradability of the substance using these results. However, taking into account a slow primary degradation (5.2 % 14CO2 of 14C-CH3-BHT) and the volatilisation of BHT (26.2 %) after 35 days, the substance could be considered as not readily biodegradable.
Executive summary:

The amounts of 14CH3- or 14C-phenyl-BHT (as an ethanol solution to give a well-suspended BHT) and activated sludge were added to the standard culture solution (100 mL) and each of the mixture was incubated aerobically by supplying CO2 free air continuously at the rate of 5 mL/min over 5-16 weeks at 25 ± 1 ºC in the dark. The 14CO2 trap was replaced weekly. BHT was biodegraded quite easily (half life 3-7 days) by activated sludge with evolution of radioactive CO2 (26-50% of the applied radiocarbon over 35-112 days). The degree of decomposition was depending on BHT load, concentrations of the applied BHT and inoculated activated sludge and duration of incubation. Volatilization should be taken into consideration in assessing biodegradation.

According to the OECD Guidance on Aquatic Toxicity Testing of Difficult Substances and Mixtures (June 2000), the concentration of the solvent in the final test medium should not exceed 0,1 mL/L. However, in this experimental work and considering the very low solubility of BHT, a series of tests were carried out with radiolabeled BHT in different concentrations together with different concentrations of activated sludge and with 0.2 mL/L ethanol in an aerated system. Two experiments were carried out adding BHT without ethanol but only one was conducted using a concentration of BHT near the water solubility limit (1 mg/L). Therefore, only the results from this experiment should be taken into account. Slow primary degradation (5.2 % 14CO2 of 14C-CH3-BHT), residual BHT in solution (10.8 %) and volatilisation of BHT (26.2 %) after 35 days of incubation is reported. In order to classify a substance a readily biodegradable, taking into account the criteria established in the CLP Regulation, in a 28-day ready biodegradation study based on carbon dioxide generation, at least a level of 60 % of theoretical maximum.degradation must be achieved. There are some results provided in this experimental work which refer to a period of 28 days using a concentration of 0.3 ppm BHT in 100 ppm activated sludge. Taking into account a degradation of 4.7 % 14CO2 of 14C-CH3-BHT and the volatilisation of BHT (23.1 %) after 28 days, the substance could be considered as not readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
A Modified MITI test according to OECD guideline 301C was conducted. However, the concentration of BHT used was well above the water solubility limit.
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Parameter:
other: BOD
Value:
4.5
Sampling time:
28 d
Validity criteria fulfilled:
not specified
Conclusions:
In a modified MITI test according to OECD guideline 301 C, an unadapted mixed microbial inoculum mineralized 4.5 % BHT within 28 days of incubation. However, the concentration of BHT used in the test was 30 mg/L which is well above the water solubility limit.
Executive summary:

In a modified MITI test according to OECD guideline 301 C, an unadapted mixed microbial inoculum mineralized 4.5 % BHT within 28 days of incubation. However, the concentration of BHT used in the test was 30 mg/L which is well above the water solubility limit.

Endpoint:
biodegradation in water: screening tests
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
accepted calculation method
Remarks:
Internationally accepted method, EPI-Suite, EPA (USA)
Principles of method if other than guideline:
QSARs estimation by EPI-Suite, EPA (USA) / BIOWIN v4.10
GLP compliance:
no
Key result
Remarks on result:
not readily biodegradable based on QSAR/QSPR prediction
Validity criteria fulfilled:
not applicable
Conclusions:
Ready Biodegradability Prediction: NO
Executive summary:

 Probability of Rapid Biodegradation (BIOWIN v4.10):

  Biowin1 (Linear Model)        :  0.4453

  Biowin2 (Non-Linear Model)    :  0.1109

 Expert Survey Biodegradation Results:

  Biowin3 (Ultimate Survey Model):  2.2695 (weeks-months)

  Biowin4 (Primary Survey Model) :  3.1941 (weeks      )

 MITI Biodegradation Probability:

  Biowin5 (MITI Linear Model)   :  0.3164

  Biowin6 (MITI Non-Linear Model):  0.1318

 Anaerobic Biodegradation Probability:

  Biowin7 (Anaerobic Linear Model): -0.7917

 Ready Biodegradability Prediction:  NO

Description of key information

Experimental results from the scientific article: According to the OECD Guidance on Aquatic Toxicity Testing of Difficult Substances and Mixtures (June 2000), the concentration of the solvent in the final test medium should not exceed 0,1 mL/L. However, in this experimental work and considering the very low solubility of BHT, a series of tests were carried out with radiolabeled BHT in different concentrations together with different concentrations of activated sludge and with 0.2 mL/L ethanol in an aerated system. Two experiments were carried out adding BHT without ethanol but only one was conducted using a concentration of BHT near the water solubility limit (1 mg/L). Therefore, only the results from this experiment should be taken into account. Slow primary degradation (5.2 % 14CO2 of 14C-CH3-BHT), residual BHT in solution (10.8 %) and volatilisation of BHT (26.2 %) after 35 days of incubation is reported. In order to classify a substance a readily biodegradable, taking into account the criteria established in the CLP Regulation, in a 28-day ready biodegradation study based on carbon dioxide generation, at least a level of 60 % of theoretical maximum.degradation must be achieved. There are some results provided in this experimental work which refer to a period of 28 days using a concentration of 0.3 ppm BHT in 100 ppm activated sludge. Taking into account a degradation of 4.7 % 14CO2 of 14C-CH3-BHT and the volatilisation of BHT (23.1 %) after 28 days, the substance could be considered as not readily biodegradable.

Based on the estimated results using EPI-Suite, the substance is considered as not readily biodegradable.

A Modified MITI test according to OECD guideline 301C was conducted. However, the concentration of BHT used in the test was 30 mg/L which is well above the water solubility limit.

Therefore, and based on the weight of evidence analysis, the substance is considered as not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Weight of evidence: Experimental results from a scientific article and estimated data using EPI-Suite.