Phenol, 4-methyl-, reaction products with dicyclopentadiene and isobutylene

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: screening tests

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 January 1997 to 21 March 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to OECD guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Inoculum was adapated to the test substance

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Deviations:

yes

Remarks:

Only pre-exposure part was conducted

Principles of method if other than guideline:

The study method was a combination of the 1992 revisions of OECD Guidelines 302B and 301B due to the low solubility of the test substance in water. The study consists of two phases: PHASE I comprises the pre-exposure of the inoculum based on the Zahn-Wellens/EMPA procedure (OECD 302B), PHASE II is the subsequent biodegradation assessment based on the CO2 evolution test (OECD 301B) as OECD 302B is not suitable for non-water-soluble substances.

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

PHASE I (PHASE II: inoculum from PHASE I was immediately used after termination, more details at end of this text box)
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): activated sludge was collected from one of the return lines at Burley Menston sewage treatment works (Yorkshire Water), a treatment works whose waste-water catchment is predominantly domestic. The activated sludge inoculum was no acclimatised or adapted to Wingstay L-HLS before exposure to the test substance in this test.
- Storage conditions: no data
- Storage length: maximum 6 hours after collection
- Preparation of inoculum for exposure:
* step 1: on arrival in the laboratory, sludge was aerated by means of compressed air supply delivered through a diffuser block
* step 2: air supply interrupted to settle solids
* step 3: siphoning of clear supernatant
* step 4: restoring original volume with dechlorinated mains water
* steps 2 to 4 were repeated once more
* step 5: measurement of suspended solids concentration by filtering a 25-mL subsample through a pre-dried and pre-weighed glass microfibre filter (Whatman GF/C); filter and retained solids were then dried by microwave oven, re-weighed and contribution made by sludge solids determined by difference.
- Concentration of sludge: no data
- Initial cell/biomass concentration: no data
- Water filtered: yes
- Type and size of filter used, if any: glass microfibre filter (Whatman GF/C)

STOCK/TEST SOLUTIONS
- Reference substance: prepared by dissolving 2.2086 g diethylene glycol in 250 mL reverse-osmosis water
- Test solution: 1 L of medium with 10 mL/L of sub-medium 1 and 1 mL/L of sub-mediums 2, 3 and 4.

TEST CONDITIONS
- Composition of medium: synthetic mineral salts medium based on reverse-osmosis water:
* sub-medium 1: 8.4996 g potassium dihydrogen phosphate (BDH), 21.7503 g dipotassium hydrogen phosphate (Aldrich), 33.4002 g disodium hydrogen pohsphate dihydrate (BDH), 0.5000 g ammonium chloride (Sigma), all dissolved in and made up to 1 L in reverse-osmosis water
* sub-medium 2: 36.4005 g calcium chloride dihydrate (Fisons), dissolved in and made up to 1 L in reverse-osmosis water
* sub-medium 3: 22.5002 g magnesium sulphate heptahydrate (Aldrich), dissolved in and made up to 1 L in reverse-osmosis water
* sub-medium 4: 0.2500 g ferric chloride hexahydrate (Sigma) plus one drop concentrated hydrochloric acid (BDH), dissolved in and made up to 1 L in reverse-osmosis water
- Additional substrate: not applicable
- Solubilising agent (type and concentration if used): not used, but due to insolubility of the test substance in water, Wingstay L-HLS flakes were ground to a fine powder with an agate pestle and mortar and weighed before addition to the medium
- Test temperature: nominal at 21 °C (controled facility), measured ranging from 20.0 to 22.9 °C
- pH: (mean ± sd) blank 7.39 ± 0.08, test vessel 7.38 ± 0.09, toxicity control 7.32 ± 0.13, reference 7.34 ± 0.14 (measured on 15 occasions in total)
- pH adjusted: no
- Dissolved oxygen concentration: (mean ± sd) blank 8.9 ± 0.2, test vessel 8.9 ± 0.2, toxicity control 8.8 ± 0.2, reference 8.9 ± 0.2 (meausred on 15 occasions in total)
- CEC (meq/100 g): no data
- Aeration of dilution water: yes
- Suspended solids concentration: 5.204 g/L
- Continuous darkness: yes
- Duration of test: 28 days

TEST SYSTEM
- Each test vessel contained 4 L of medium with 154 mL of sludge (inoculum), i.e. equivalent of 800 mg dry solids.
- One replicate per treatment (blank, test substance only, reference substance and toxicity control (i.e. test and reference substance))

SAMPLING for DOC analysis
- Sampling frequency: every two to three days
- Sampling method: 50 mL of was withdrawn from the vessels and filtered through 0.2 µm pore inorganic membrane filters (Anodisc 47, Whatman); filtrates were collected for analysis.
- Sterility check if applicable: not applicable
- Sample storage before analysis: refrigerated when maxium storage time was seven days, frozen when storage time was more than 7 days (thawed immediately prior to analysis)

DOSING OF TEST, CONTROL, REFERENCE AND BLANK VESSELS
- Test vessel: weighed quantity of Wingstay L-HLS powder (0.2364 g) was flushed with mineral salts medium directly to the test vessel, nominal concentration of 50 mg organic carbon per litre, or 59 mg/L as Wingstay L-HLS
- Inoculum blank: yes
- Abiotic sterile control: not applicable
- Toxicity control: weighed quantity of Wingstay L-HLS powder ( 0.2358 g) was flushed with mineral salts medium directly to the toxiticy control vessel, nominal concentration of 50 mg organic carbon per litre, or 59 mg/L as Wingstay L-HLS. In addition, 50 mL of reference stock solution was added, providing a nominal diethylene glycol concentration corresponding to 50 mg organic carbon/L.
- Reference control: 50 mL of reference stock solution was added, providing a nominal diethylene glycol concentration corresponding to 50 mg organic carbon/L.

CALCULATIONS
Biodegradation expressed in terms of percentage DOC elimination was calculated by applying the formula:
% biodegradation = 1 - [(DOCTt - DOCBt)/(DOCT3h - DOCB3h)] x 100
with DOCTt = DOC in test vessel at time t
DOCBt = DOC in blank vessel at time t
DOCT3h = DOC in test vessel after the first 3h
DOCB3h = DOC in blank vessel after the first 3h

RESULTS of DOC elimination
- see Overall remarks/Attachments for Illustration: Percentage DOC elimination in the Zahn-Wellens/EMPA test
- The mean DOC elimination measured in the test vessel was 1.1 mg C/L, and this confirmed that the test substance is not soluble in water to any significant extent.

PHASE II
- Source of inoculum: sludge harvested from PHASE I; recovery achieved by stopping aeration to settle sludge solids, followed by siphoning off cleared supernatant
- Suspended solids concentration: determined by filtering a 25-mL subsample (procedure described above): 3.184 g/L
- Speks of residual powdered test substance were visible in the transferred inoculum, but would have been distributed evenly between blank and test vessel in phase II

Duration of test (contact time):

28 d

Initial conc.:

17.6 - ca. 17.8 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Remarks:

, cumulative recovered yield expressed as percentage of theoretical and calculated from the carbon content of the test substance

Details on study design:

PHASE II
TEST CONDITIONS
- Composition of medium: 5 L of medium with 10 mL/L of sub-medium 1 and 1 mL/L of sub-mediums 2, 3 and 4 (detailed composition see section "Details on inoculum") inoculated with the entire residual recovered sludge volume (97 mL)
- Each test vessel was purged overnight with CO2-free air to drive out dissolved CO2 before introducing the test substance.
- Additional substrate: not applicable
- Solubilising agent (type and concentration if used): not used, but due to insolubility of the test substance in water, Wingstay L-HLS flakes were ground to a fine powder with an agate pestle and mortar and weighed before addition to the medium
- Test temperature: nominal at 21 °C (controled facility), measured ranging from 21.4 to 24.0 °C
- pH: measured only in the blanks at the start of the incubation, during the incubation test vessels were not analysed for pH to prevent adherence of undissolved test substance to the pH electrode; all vessels were analyzed on day 28 immediately before termination. Results see Table 2.
- pH adjusted: no
- CEC (meq/100 g): no data
- Aeration of dilution water: yes, nominally CO2-free 'control air' (Air products), fed from cylinders; as an added precaution, the air flow passed through a column packed with 'Carbosorb AS' (Merck), a self-indicating, articifical silicate CO2-absorber
- Suspended solids concentration: nominal concentration of 21 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: test vessels contained 1 L of medium and 3 L of reverse-osmosis water and weighed quantities of Wingstay L-HLS powder (0.0535 g and 0.0529 g) which were flushed with mineral salts medium directly to the test vessel, providing nominal concentrations of 15 mg organic carbon per litre, or 17.6-17.8 mg/L as Wingstay L-HLS
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: air flow regulated in two stages; initial coarse control provided by air taps and air flow to each vessel regulated by individual needle valves
- Method used to create anaerobic conditions: not applicable
- Measuring equipment for air flow: bubble flow meter and stop-watch, at intervals not exceeding three days
- Test performed in closed vessels due to significant volatility of test substance: not applicable
- Test performed in open system: no data
- Details of trap for CO2 and volatile organics if used: series of dedicated CO2-scrubbers containing a barium hydroxide (Ba(OH)2) solution

SAMPLING
- Sampling frequency: on days 2, 4, 6, 8, 10, 15, 20, 25 and 28
- Sampling method: see section Details on analytical methods
- Sterility check if applicable: not applicable
- Sample storage before analysis: no data

CALCULATIONS:
- Biodegradation expressed in terms of percentage theoretical CO2 yield was calculated by applying the formula:
biodegradation = cumulative mg CO2 produced at time t/165 x 100
- a factor of five was used to convert titres fro 20 mL samples to values for the entire 100 mL wash-bottle contents and a factor of 1.1 was applied to convert volume-corrected titres to mg CO2.
- theoretical yields for the quantities of Wingstay L-HLS applied in the test vessels were 165 mg CO2

Reference substance:

diethylene glycol

Parameter:

% degradation (CO2 evolution)

Value:

1

Sampling time:

28 d

Details on results:

See Table 3 for results of all CO2 measurements.
These data are below the 10 % level generally regarded as representing the threshold of significant degradation, and below the 20 % level required to give convincing evidence for inherent biodegradability.

Results with reference substance:

- DOC removal was almost complete (94 %) by day 7 (see Table 1) in the reference vessel
- DOC elimination in toxicity control followed same pattern as described for the reference vessel
Maximum divergence from contemperary DOC elimination in the reference vessel was 6 % on Day 27, but this is not considered significant. Therefore, it may be concluded that the presence of Wingstay L-HLS in the toxicity control did not impact upon the degradation of diethylene glycol.

Table 1: Phase I: Biodegradation as percentage DOC elimination

Elapsed time, days	Toxicity control	Reference substance
2	0	4
4	1	6
7	96	94
9	99	100
13	99	100
16	99	100
20	97	96
27	92	98
28	99	100

Table 2: Phase II: Initial and final pH measurements

	Blank		Wingstay L-HLS
Elapsed time, days	replicate 1	replicate 2	replicate 1	replicate 2
0	7.76	7.75	no data	no data
28	7.75	7.82	7.76	7.72

Table 3: Phase II: Biodegradation as percentage theoretical CO2 yield

	Wingstay L-HLS
Elapsed time, days	replicate 1	replicate 2
2	0	0
4	0	0
6	0	0
8	1	0
10	1	0
15	1	0
20	1	0
25	1	1
28	1	1

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

Reliable study (Klimisch 1) investigating the biodegradation of the water-insoluble substance Wingstay L-HLS. The pre-adaptation of the inoculum demonstrated that the applied concentration of Wingstay L-HLS did not adversely affect the sludge micro-organisms. This is an important indication that the lack of biodegradation is due to the persistent characteristics of the substance and not to failure of the test system. The fact that the test substance was not inherently biodegradable also implies that the substance will not be ready biodegradable.

Description of key information

1) The key study conducted by Covance (1998) combined the guidelines for ready biodegradability (OECD301B) and inherent biodegradability (OECD 302B), by first incubating the registered substance with the inoculum (non-adapted domestic activated sludge) in order to adapt it to the test substance. Subsequently, the CO2 evolution method was used to assess the potential inherent biodegradability of

the test substance. Based on the results of this study the registered substance did not show any inherent biodegradation and was therfore considerd to be not readily biodegradable under the conditions of the test.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

As the substance is not soluble in water, the OECD 302B method is not suitable. Therefore the biodegradation of the substance was assessed with the CO2 evolution method (following OECD 301B),

which is suitable for non-soluble substances. This was done after adapting the inoculum (following OECD 302B). Based on the results of this study the registered substance did not show any inherent

biodegradation and was therfore considerd to be not readily biodegradable under the conditions of the test. Even though no biodegradation has been observed, it cannot be disproved beyond reasonable doubt that the substance is not readily biodegradable as lack of biodegradation observed in the biodegradation test may be due to lack of bioavailability due to the poor water solubility of the substance. The registered substance is made to function as an anti-oxidant. It can thus be argued that the substance may abiotically degrade in the environment. However, its transformation products have not been assessed, so no definite conclusions can be drawn at the moment and further screening tests to fully understand the biodegradation of the test material and its main components are needed.