Methyl 2-naphthyl ether

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental result using OECD guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

other: Mixed inoculum

Details on inoculum:

Mixed Inoculum Preparation: Polyseed were used for mixed inoculum. One capsule of Polyseed were added in 500 ml of DI water to make up the solution. Afterwards 1 hour of stirring were given for proper mixing of mixed inoculum. This gave the bacterial count as 10E7 to 10E8 CFU/ml. At the regular interval microbial plating was also performed on agar to confirm the vitality and CFU count of microorganism.

Duration of test (contact time):

28 d

Initial conc.:

4 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: OECD mineral medium was used for the study
- Test temperature: 20°C
- Continuous darkness: Yes
- Other: The water used in this study is deionized water.

TEST SYSTEM
- Culturing apparatus: The apparatus used in this study is BOD bottles; with glass stoppers (125 ml), BOD incubator & oxygen electrode and meter.

CONTROL AND BLANK SYSTEM
- Inoculum blank: it contains only test inoculum
- Procedure control: contains reference compound and inoculum

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

50.38

Sampling time:

28 d

Remarks on result:

other: Other details not known

Details on results:

The oxygen consumed by the test systems was corrected for oxygen consumption occurring in the blank test systems.The BOD Values (mgO2/mg) and percent biodegradation results for each test system arereported in Tables 2 and 3, respectively. The BOD28 value of test chemical was observed to be 1.32 mgO2/mg. ThOD was determined by calculation as 2.62 mgO2/mg. % Degradation was calculated using the values of BOD and ThOD for test item and was found to be 50.38% at 20 ± 1°C. The % degradation of procedure control (reference item) was also calculated using the values of BOD & ThOD and was determined to be 73.49% at 20 ± 1°C. Degradation of Sodium Benzoate exceeds 33.13% on 7 days & 40.36% on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid.

Results with reference substance:

Degradation of Sodium Benzoate exceeds 33.13% on 7 days & 40.36% on 14th day. The % degradation of procedure control (reference item) was also calculated using the values of BOD & ThOD and was determined to be 73.49% at 20 ± 1°C.

 TABLE: D.O Values(mg/L)

 

No. of Days	Inoculum Blank (control)	Test Suspension	Procedure Control (Reference Item)
0	6.6	6.5	6.6
7	6.4	4.5	4.2
14	6.2	3.2	3.5
21	6.1	1.5	2.2
28	0	0.6	1.1

 

 

Table: BOD values (mgO2/mg)

No. of Days	Test Suspension	Procedure Control (Reference Item)
0	0	0
7	0.45	0.55
14	0.72	0.67
21	1.12	0.97
28	1.32	1.22

 

 

Table: PERCENT BIODEGRDATION RESULTS

No. of Days	Test Suspension	Procedure Control (Reference Item)
0	0%	0%
7	17.17%	33.13%
14	27.48%	40.36%
21	42.74%	58.43%
28	50.38%	73.49%

 

 

Table: BOD28, THOD AND % BIODEGRADATION VALUES

 

Method details	BOD28 (mgO2/mg)	ThOD (mgO2/mg)	% Biodegradation
Test Item	1.32	2.62	50.38
Reference Item	1.22	1.66	73.49

 

 

 

TABLE: pH OF CLOSED BOTTLE TEST

 

No. of Days	Inoculum Blank (control)	Test Suspension	Procedure Control (Reference Item)
0	6.8	7.1	6.9
7	6.5	6.7	6.8
14	6.4	6.5	6.6
21	6.1	6.4	6.2
28	5.8	6.2	6

 

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable, fulfilling specific criteria

Conclusions:

The test chemical undergoes 50.38% biodegradation after 28 days in the test condition. Thus, the test chemical was considered to be inherently biodegradable in nature.

Executive summary:

Biodegradation study was conducted for 28 -days following the OECD guideline 301 D for determining the ready biodegradability of the test chemical. The test system included control, test item and reference item. Polyseed were used as a test inoculum. The concentration of test and reference item ( Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was 32 ml/l. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test item and reference item. The % degradation of procedure control (reference item) was also calculated using the values of BOD & ThOD and was determined to be 73.49% at 20 ± 1°C. Degradation of Sodium Benzoate exceeds 33.13% on 7 days & 40.36% on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid. The BOD28 value of test chemical was observed to be 1.32 mgO2/mg. ThOD was calculated as 2.62 mgO2/mg. Accordingly, the % degradation of the test chemical after 28 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 50.38%. Based on the results, the test chemical, under the test conditions was considered to be inherently biodegradable in water.

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Description of key information

Biodegradation study was conducted for 28 -days following the OECD guideline 301 D for determining the ready biodegradability of the test chemical (Experimental study report, 2017). The test system included control, test item and reference item. Polyseed were used as a test inoculum. The concentration of test and reference item ( Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was 32 ml/l. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test item and reference item. The % degradation of procedure control (reference item) was also calculated using the values of BOD & ThOD and was determined to be 73.49% at 20 ± 1°C. Degradation of Sodium Benzoate exceeds 33.13% on 7 days & 40.36% on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid. The BOD28 value of test chemical was observed to be 1.32 mgO2/mg. ThOD was calculated as 2.62 mgO2/mg. Accordingly, the % degradation of the test chemical after 28 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 50.38%. Based on the results, the test chemical, under the test conditions was considered to be inherently biodegradable in water.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Additional information

Various experimental studies of the test chemical were reviewed for the biodegradation end point which are summarized as below:

 

In an experimental key study from study report (2017), biodegradation study was conducted for 28 -days following the OECD guideline 301 D for determining the ready biodegradability of the test chemical. The test system included control, test item and reference item. Polyseed were used as a test inoculum. The concentration of test and reference item ( Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was 32 ml/l. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test item and reference item. The % degradation of procedure control (reference item) was also calculated using the values of BOD & ThOD and was determined to be 73.49% at 20 ± 1°C. Degradation of Sodium Benzoate exceeds 33.13% on 7 days & 40.36% on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid. The BOD28 value of test chemical was observed to be 1.32 mgO2/mg. ThOD was calculated as 2.62 mgO2/mg. Accordingly, the % degradation of the test chemical after 28 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 50.38%. Based on the results, the test chemical, under the test conditions was considered to be inherently biodegradable in water.

 

Another biodegradation study of test substance was carried out for 96 hrs using various bacterial organisms each containing different naphthalene degrading enzyme (NAH) systems in plasmids (Jeffrey D. Leblond et al, 2001). Following strains were used Pseudomonas fluorescens 5R and 5RL, Pseudomonas spp. strain DFC49 and DFC50, Pseudomonas putida PpG7 and E. coli DH5α, respectively.The purity of test substance used in the study is greater than 95%. The analysis of test chemical by gas chromatography (GC)/ mass spectrometry was carried out. ThePAH mixture containing the test substance under study was prepared fresh prior to each experiment by dissolving 0.05 g of each compound together in 5 ml of acetone. This concentrated solution was then added to 1 l of minimal salts medium (pH 7), and allowed to sit in the dark for 3 days in order to achieve full saturation. After equilibration, 5 ml aliquots of this solution were filltered through a 0.2µm PTFE syringe top filter and added to sterilized 12 ml screw cap tubes with teflon-coated caps. The other bacterial strains utilized in the biodegradation experiment were pre-grown overnight to late-log phase in a yeast extract peptone- sodium succinate-sodium salicylate (YEPSS) medium. The bacterial strain in use was centrifuged and washed three times in minimal salts medium, and then re-suspended in 20 ml of minimal salts medium to achieve a 100-fold concentration of cells.50 µl of 100-fold concentrated solution of bacterial cells were added to tubes containing the polyaromatic hydrocarbons (PAHs) mixture (which contains the test substance ) to achieve a cell density of 108cells/ml. Serial dilutions and plating on yeast extract-peptone-glucose (YEPG) medium were performed approximately every 24 h. On the average, the number of colony forming units (cfu) was maintained at 108cells/ml through the 48 h time point. From 48 to 96 h, the cfu concentration dropped one order of magnitude. The tubes were then placed on a  Glass-col rotary shaker at room temperature. Triplicate samples were taken every half an hour from time 0 up to 7 h by adding 5 ml of hexane to the appropriate sacrificed tubes and then shaking horizontally for 2 h at 150 rpm. After this time, approximately 2 ml of the hexane phase was taken from the tube, crimp-sealed in an autosampler vial with a teflon-lined cap, and then stored at  -20°C until analysis. Samples were also usually taken at 12, 24, 48, 72, and 96 h during the experiment. Negative controls, killed with concentrated sulfuric acid prior to time zero, were treated identically. Gas chromatography (GC) analysis of the hexane extracts were carried out. GC analysis of hexane extracts showed 100% disappearance of test substance by test organism Pseudomonas fluorescens 5R within 72 hrs. The first order rate constant and r2value was also noted and determined to be 0.05 h-1and 0.822, respectively. Thus, indicating that the test substance to be readily biodegradable.

 

In a supporting study from peer reviewed journal (EFFREY D. LEBLOND et al, 2000), biodegradation experiment of test chemical was carried out for 18hrs using Pseudomonas fluorescens 5RL.The purity of test substance used is greater than 95% indicating that no further purification was necessary. Bacterial culture used for the study is Pseudomonas fluorescens 5RL.Strain 5RL was pre-grown to late log phase at 28ᵒC and 150 rpm in 100 ml of a yeast extract–peptone– glucose medium amended with 14 mg/L of tetracycline. Ten milliliters of cells was then transferred to two 4.0-L Erlenmeyer flasks, each of which contained 1 L of a yeast extract– peptone–salicylic acid–succinate medium with the same concentration of tetracycline. Cells were harvested in the exponential phase of growth by centrifugation for 10 min at 22,095gand 4ᵒC. The cells were washed three times and resuspended in 200 ml of 50 mM sodium phosphate buffer (pH 7.0) to an approximate concentration of 1.0 g of cells per 100 ml.The bacterial cells which were washed in 200 ml of 50 mM sodium phosphate buffer was then dividedin half into two 500- ml Erlenmeyer flasks. Into one flask was added 20 mg of substrate dissolved in 0.5 ml of N,N-dimethylformamide. The negative control consisted of addingN,N-dimethylformamide to 100 ml of cells. Similar negative control experiments were performed usingEscherichia colicontaining pUTK202 grown on Luria-Bertani broth in the presence of 50 mg/L of ampicillin. All flasks were then incubated at 28ᵒC and 150 rpm for approximately 18 h. After centrifugation, the cell-free suspensions were extracted with ethyl acetate (neutral extract; three 100-ml volumes). The aqueous layer was then acidified (pH < 2.0) with concentrated H2SO4 and extracted with ethyl acetate (acid extract). Both the neutral and acid ethyl acetate extracts were dried over anhydrous sodium sulfate, and the solvent was removed in vacuo at 30ᵒC. The residues were dissolved in 2.0 ml of acetone and then prefiltered through a 0.2-µm-pore-size polytetrafluoroethylene filter before analysis.Gas chromatography (GC) analysis of neutral extracts were carried out.GC-MS analysis of neutral extracts showed that the test substance undergoes transformation by Pseudomonas fluorescens 5RL and transformation product was determined to be 4-methoxysalicylic acid. Thus, based on this, test chemical can considered to be biodegradable in water.

 

On the basis of the study result conducted as per the OECD guideline 301 D, it is concluded that the test chemical was considered to be inherently biodegradable in water.