Methyl 2-naphthyl ether

Endpoint summary

Description of key information

Hydrolysis

In accordance with column 2 Annex IX and Annex XIII of REACH regulation, study does not need to be conducted since the substance has a low potential for bioaccumulation (logKow <3.5).
Additionally, the substance undergoes a degradation of 89% at 30 days as per OECD TG 309 OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) under aerobic conditions, thus considered as not persistent in water and thereby has a low potential to cross biological membranes of test species.

 

Biodegradation in water

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.

 

Biodegradation in water and sediment

Primary biodegradation in surface water from a Danish stream was determined by compound specified analysis. The study was performed following the modified OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) under aerobic conditions. Natural water (stream of good ecological quality and without known point sources close to sampling point) sampled from Gudenåen (DK) 16th of June 2020. WGS84: 56°6'23.2"N 9°43'17.4"E at a dept of 5 -20 cm below surface was used as a surface water. The water was quite clear with a slight light brown hint. Total suspended solids was 2.0 mg/L, total dissolved solids was 210 mg/L. No pre-treatment was done of the natural water. Water kept at 20 ± 1.5°C during transport to lab (4 hours), then stored at 12°C until test setup within 30 hours of sampling. The temperature and pH at the time of collection of the sample was 20.0°C and 6.5 pH. Biomass i.e., plate count at 24 and 72 hr was 36 CFU/ml and 1281 CFU/ml, respectively. Water was not filtered. Dissolved non-volatile organic carbon (NVOC) was measured instead of DOC/TOC and was 4.4 mg/L. Study was performed using 20 mL amber headspace closed vials. A large no. of biotic test systems (15 -18 replicates) consisting of 14.5 ml surface water and 0.5 ml stock solution was prepared test vials. Stock solution was prepared in water by equilibrium partioning from a pre-loaded silicone rod (passive dosing). This method for preparing stock solutions avoids the need for solvents for spiking. A similar number of abiotic control test systems were prepared using 14.5 ml MilliQ water and 0.5 ml stock solution. Blanks were prepared with 15 ml MilliQ water. Aerobic conditions created by using aerobic natural water (initial oxygen content 9.0 mg/L at 20 °C)  and incubating using a headspace of 5 mL with a low test substance concentration of 0.3 mg/l. Test was conducted at a temperature of 12°C and pH of 6.5 under continuous darkness with conductivity of 334 µS/cm while rolling. pH of the test medium (natural water) was not adjusted. No aeration was provided during the study. Suspended solid concentrations uded in the study was 2.0 mg/l. At 5 -6 time-points, 3 biotic and 3 abiotic test systems were sacrificed for analysis (day2, 6, 14, 21, 27 and 30). Tests systems were put directly on the autosampler of the GCMS without storage. Blank test systems were prepared and incubated with biotic and abiotic test systems and analyzed at each time-point (signal well below the 3% dilution of initial test concentration). Analysis was carried out using direct immersion solid phase microextraction coupled to GC-MS. Carryover was < 1.0% and since test systems were analyzed alternating biotic and abiotic test systems this constitutes the detection limit in this test. Biodegradation was then evaluated by the ratio between peak areas in the biotic and abiotic test systems. Triplicate ratios of biotic/abiotic test system peak areas were calculated for each time point and used in Graph-pad Prism 8.4.3 to fit the first order degradation model with lag phase. The DT50 of the test chemical was evaluated to be 26 days at 12°C. Primary percentage degradation of the test chemical was determined to be 89% at 30 days. Thus, on the basis of results, test chemical was considered to be not persistent in surface water.

 

Biodegradation in soil

The half-life period of test chemical in soil was estimated using Level III Fugacity Model by EPI Suite version 4.1 estimation database (2017). If released into the environment, 79.9 % of the chemical will partition into soil according to the Mackay fugacity model level III. The half-life period of test chemical in soil is estimated to be  30 days (720 hrs). Based on this half-life value of test chemical, it is concluded that the chemical is not persistent in the soil environment and the exposure risk to soil dwelling animals is moderate to low.

 

Bioaccumulation: aquatic / sediment

BCFBAF model (v3.01) of Estimation Programs Interface (2018) was used to predict the bioconcentration factor (BCF) of test chemical . The bioconcentration factor (BCF) of test chemical was estimated to be 90.47 L/kg whole body w.w (at 25 deg C) which does not exceed the bio concentration threshold of 2000, indicating that the chemical is not expected to bioaccumulate in the food chain.

 

Adsorption / desorption

The adsorption coefficient Koc in soil and in sewage sludge of test chemical was determined by the High Performance Liquid Chromatographic method according to OECD Guideline No. 121 for testing of Chemicals. The solutions of the test substance and reference substances were prepared in methanol solvent. For the preparation of test solution 10.37 mg of the test item was weighed and transferred into a 10 mL volumetric flask, dissolved and volume was made up to the mark with methanol. 100µl of this solution was pipetted out into vial and 900µl of methanol added to it was shaken well. The concentration of the test chemical solution was 103.7mg/L.  After column saturation with the mobile phase, sequential analysis of thiourea (for dead time: t0), reference substances and test chemical were performed on HPLC at 254 nm. In HPLC system, thiourea was injected first, the reference substances were injected, followed by the test chemical solution. The 10ml volume of each was introduced in the column by using autosampler. All the HPLC runs were performed in duplicates. Retention time tR was measured to calculate the capacity factors k', it was then converted in log to obtain log k'. A standard curve between log k versus log Koc was plotted to obtain the value of log Koc of the test chemical. Reference substance taken for study includes Acetanilide, Atrazine, Methyl Benzoate, Naphthalene , Phenanthrene and Acetophenone having log Koc value ranging from 1.25 to 4.09 dimensionless. The Adsorption Coefficient of test chemical was determined as per the HPLC method (OECD Guideline-121). The Log Koc value was determined to be 3.06 dimensionless at 30°C. This log Koc value indicates that the test chemical has a moderate sorption to soil and sediment and therefore have slow migration potential to ground water.

Additional information

Hydrolysis

In accordance with column 2 Annex IX and Annex XIII of REACH regulation, study does not need to be conducted since the substance has a low potential for bioaccumulation (logKow <3.5).
Additionally, the substance undergoes a degradation of 89% at 30 days as per OECD TG 309 OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) under aerobic conditions, thus considered as not persistent in water and thereby has a low potential to cross biological membranes of test species.

 

Biodegradation in water

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.

 

Biodegradation in water and sediment

Studies of biodegradation were evaluated for determing the persistence of test chemical. The studies are reported below:

 

In an experimental study, primary biodegradation in surface water from a Danish stream was determined by compound specified analysis. The study was performed following the modified OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) under aerobic conditions. Natural water (stream of good ecological quality and without known point sources close to sampling point) sampled from Gudenåen (DK) 16th of June 2020. WGS84: 56°6'23.2"N 9°43'17.4"E at a dept of 5 -20 cm below surface was used as a surface water. The water was quite clear with a slight light brown hint. Total suspended solids was 2.0 mg/L, total dissolved solids was 210 mg/L. No pre-treatment was done of the natural water. Water kept at 20 ± 1.5°C during transport to lab (4 hours), then stored at 12°C until test setup within 30 hours of sampling. The temperature and pH at the time of collection of the sample was 20.0°C and 6.5 pH. Biomass i.e., plate count at 24 and 72 hr was 36 CFU/ml and 1281 CFU/ml, respectively. Water was not filtered. Dissolved non-volatile organic carbon (NVOC) was measured instead of DOC/TOC and was 4.4 mg/L. Study was performed using 20 mL amber headspace closed vials. A large no. of biotic test systems (15 -18 replicates) consisting of 14.5 ml surface water and 0.5 ml stock solution was prepared test vials. Stock solution was prepared in water by equilibrium partioning from a pre-loaded silicone rod (passive dosing). This method for preparing stock solutions avoids the need for solvents for spiking. A similar number of abiotic control test systems were prepared using 14.5 ml MilliQ water and 0.5 ml stock solution. Blanks were prepared with 15 ml MilliQ water. Aerobic conditions created by using aerobic natural water (initial oxygen content 9.0 mg/L at 20 °C)  and incubating using a headspace of 5 mL with a low test substance concentration of 0.3 mg/l. Test was conducted at a temperature of 12°C and pH of 6.5 under continuous darkness with conductivity of 334 µS/cm while rolling. pH of the test medium (natural water) was not adjusted. No aeration was provided during the study. Suspended solid concentrations uded in the study was 2.0 mg/l. At 5 -6 time-points, 3 biotic and 3 abiotic test systems were sacrificed for analysis (day2, 6, 14, 21, 27 and 30). Tests systems were put directly on the autosampler of the GCMS without storage. Blank test systems were prepared and incubated with biotic and abiotic test systems and analyzed at each time-point (signal well below the 3% dilution of initial test concentration). Analysis was carried out using direct immersion solid phase microextraction coupled to GC-MS. Carryover was < 1.0% and since test systems were analyzed alternating biotic and abiotic test systems this constitutes the detection limit in this test. Biodegradation was then evaluated by the ratio between peak areas in the biotic and abiotic test systems. Triplicate ratios of biotic/abiotic test system peak areas were calculated for each time point and used in Graph-pad Prism 8.4.3 to fit the first order degradation model with lag phase. The DT50 of the test chemical was evaluated to be 26 days at 12°C. Primary percentage degradation of the test chemical was determined to be 89% at 30 days. Thus, on the basis of results, test chemical was considered to be not persistent in surface water.

 

For the test chemical, Estimation Programs Interface (2017) prediction model was run to predict the half-life in sediment for the test chemical. The half-life period of test chemical in sediment is estimated to be 135 days (3240 hrs). However, as the percentage release of test chemical into the sediment is less than 1% (i.e, reported as 0.673%), indicates that test chemical is not persistent in sediment.

 

Thus, test chemical was considered to be not persistent.

 

Biodegradation in soil

The half-life period of test chemical in soil was estimated using Level III Fugacity Model by EPI Suite version 4.1 estimation database (2017). If released into the environment, 79.9 % of the chemical will partition into soil according to the Mackay fugacity model level III. The half-life period of test chemical in soil is estimated to be  30 days (720 hrs). Based on this half-life value of test chemical, it is concluded that the chemical is not persistent in the soil environment and the exposure risk to soil dwelling animals is moderate to low.

 

On the basis of available information, the test chemical is considered to be inherently biodegradable in water.

 

Bioaccumulation: aquatic / sediment

Various predicted data for the test chemical and supporting weight of evidence studies for its structurally and functionally similar read across substance were reviewed for the bioaccumulation end point which are summarized as below:

 

In aprediction done using the BCFBAF Program(v3.01) of Estimation Programs Interface (2018) prediction program was used to predict the bioconcentration factor (BCF) of test chemical. The bioconcentration factor (BCF) of test chemical was estimated to be 90.47 L/kg whole body w.w (at 25 deg C).

 

Another prediction was done by using OECD QSAR toolbox version 3.4 (2019) to estimate the bioaccumulation value (BCF) of test chemical in fish. The BCF value was estimated to be 366 dimensionless whole body w.w.

 

In an another prediction done by using Bio-concentration Factor (v12.1.0.50374) moduleACD (Advanced Chemistry Development)/I-Lab predictive module, 2017), thebio-concentration factor over the entire pH scale (pH 1 -14) of the test chemical was estimated to be 212.

 

From CompTox Chemistry Dashboard using OPERA (OPEn (quantitative) structure-activity Relationship Application)  V1.02 model in which calculation based on PaDEL descriptors (calculate molecular descriptors and fingerprints of chemical)  the bioaccumulation i.e BCF for test chemical was estimated to be 293 dimensionless . The predicted BCF result based on the 5 OECD principles.

 

In a supporting weight of evidence study from authoritative database (2017) and secondary source, bioaccumulation study was conducted on test organism Cyprinus carpio for 8 weeks for evaluating the bioconcentration factor (BCF value) of test chemical. The study was performed according to other guideline "Bioaccumulation test of a chemical substance in fish or shellfish" provided in "the Notice on the Test Method Concerning New Chemical Substances", respectively. Cyprinus carpio was used as a test organism for the study. Test chemical nominal conc. used for the study were 0.05 mg/l and 0.005 mg/l, respectively. Test chemical solution was prepared in HCO-40. Analytical method involve the recovery ratio: Test water : 1st concentration area : 87.3 %, 2nd concentration area : 90.0 %, Fish : 91.0 %, - Limit of detection : Test water: 1st concentration area : 2.4 ng/ml, 2nd concentration area : 0.23 ng/ml, Fish : 69 ng/g. Range finding study involve theLC50(48h) 11.1 mg/Lon Rice fish (Oryzias latipes). Lipid content of the test organism Cyprinus carpio was determined to be 4.1% at the start of exposure. The bioconcentration factor (BCF value) of substance on Cyprinus carpio was determined to be in the range of 431-1180 L/Kg at a conc. of 0.05 mg/l and 398-873 L/Kg at a conc. of 0.005 mg/l, respectively, which does not exceed the bioconcentration threshold of 2000, indicating that the test chemical is not expected to bioaccumulate in the food chain.

 

For the test chemical,bioaccumulation study was conducted on test organism Cyprinus carpio for 6 weeks for evaluating the bioconcentration factor (BCF value) of test chemical. The study was performed according to OECD Guideline 305 C (Bioaccumulation: Test for the Degree of Bioconcentration in Fish) and other guideline "Bioaccumulation test of a chemical substance in fish or shellfish" provided in "the Notice on the Test Method Concerning New Chemical Substances", respectively at 25°C. Cyprinus carpio was used as a test organism for the study. Test chemical nominal conc. used for the study were 0.1mg/l and 0.01 mg/l, respectively. Test chemical solution was prepared in HCO-20. Analytical method involve the recovery ratio: Test water : 96.7 %, Fish : 88.5 %, - Limit of quantitation : Test water : 1st concentration area : 2.7 microg/L, 2nd concentration area : 0.27 microg/L, Fish : 33 ng/g. Range finding study involve the LC50(48h) ≥ 26 mg/Lon Rice fish (Oryzias latipes). Lipid content of the test organism Cyprinus carpio was determined to be 4.0% at the start of exposure. The bioconcentration factor (BCF value) of test chemical on Cyprinus carpio was determined to be in the range of 6.1 to 63 L/Kg at a conc. of 0.1 mg/l and 5.9 to 23 L/Kg at a conc. of 0.01 mg/l, respectively, which does not exceed the bioconcentration threshold of 2000, indicating that the test chemical is not expected to bioaccumulate in the food chain.

 

On the basis of above results of the test chemical, it can be concluded that the BCF value of test chemical was evaluated to be ranges from 5.9 to 1180, respectively,which does not exceed the bioconcentration threshold of 2000, indicating that the test chemical is not expected to bioaccumulate in the food chain.

 

Adsorption / desorption

The adsorption coefficient Koc in soil and in sewage sludge of test chemical was determined by the High Performance Liquid Chromatographic method according to OECD Guideline No. 121 for testing of Chemicals. The solutions of the test substance and reference substances were prepared in methanol solvent. For the preparation of test solution 10.37 mg of the test item was weighed and transferred into a 10 mL volumetric flask, dissolved and volume was made up to the mark with methanol. 100µl of this solution was pipetted out into vial and 900µl of methanol added to it was shaken well. The concentration of the test chemical solution was 103.7mg/L.  After column saturation with the mobile phase, sequential analysis of thiourea (for dead time: t0), reference substances and test chemical were performed on HPLC at 254 nm. In HPLC system, thiourea was injected first, the reference substances were injected, followed by the test chemical solution. The 10ml volume of each was introduced in the column by using autosampler. All the HPLC runs were performed in duplicates. Retention time tR was measured to calculate the capacity factors k', it was then converted in log to obtain log k'. A standard curve between log k versus log Koc was plotted to obtain the value of log Koc of the test chemical. Reference substance taken for study includes Acetanilide, Atrazine, Methyl Benzoate, Naphthalene , Phenanthrene and Acetophenone having log Koc value ranging from 1.25 to 4.09 dimensionless. The Adsorption Coefficient of test chemical was determined as per the HPLC method (OECD Guideline-121). The Log Koc value was determined to be 3.06 dimensionless at 30°C. This log Koc value indicates that the test chemical has a moderate sorption to soil and sediment and therefore have slow migration potential to ground water.