2-ethoxy-4-(hydroxymethyl)phenol

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 4 September 2014 Experimental completion date: 13 November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-ethoxy-4-(hydroxymethyl)phenol

EC Number:

674-192-5

Cas Number:

4912-58-7

Molecular formula:

C9H12O3

IUPAC Name:

2-ethoxy-4-(hydroxymethyl)phenol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Lot/batch No.of test material: S4C0002
- Purity of test item: 99.67%
- Impurity: Unknown: 0.33%
- Chemical name: 2-Ethoxy-4-(hydroxymethyl)phenol
- Another name: NACET00470
- CAS number: 4912-58-7
- Appearance: White powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in a dark place at room temperature.
- Stability under test conditions: Stable in water
- Solubility and stability of the test substance in the solvent/vehicle:
Solubility to solvent:
Dimethylsulfoxide: equal to or > 50 g/L
Acetone: equal to or > 50 g/L

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Remarks:

Prepared and controlled according to the test method.

Details on inoculum:

- Source of inoculum/activated sludge: On-site sludge sampling was carried out at 10 locations in Japan (samples were from surface water and surface soil of rivers, lakes, and inland sea; return sludge from sewage plants).
The activated sludge, which was cultivated for 21.5 hours after the synthetic sludge was added, was used. The synthetic sludge was prepared according to the following method: glucose, peptone and potassium dihydrogenphosphate were dissolved in purified water and the pH of the solution was adjusted to 7.0 +/- 1.0.

Duration of test (contact time):

28 d

Details on study design:

TEST CONDITIONS
Prepartions for test
Decision of additive amount of activated sludge
Additive amount of activated sludge in the test vessel was 2.74 mL on the basis of the concentration of suspended solid in the activated sludge which was determined by the following methods:
Method: In accordance with Japanese Industrial Standards (JIS) K 0102-2008 Section 14.1
Date: September 22, 2014
Result: 3280 mg/L

Preparation of basal culture medium
The basal culture medium (4 L) was prepared at the same proportion as the following method:
Purified water was added to each 3 mL aliquot of solutions A, B, C and D in order to prepare 1 L of solution. The pH of this solution was then adjusted to 7.0.

Preparation of test solutions
The following test solutions were prepared and incubated. Measurement of pH of the test solutions was not performed at the preparation in this test because the test item was not immediately dissolved at the preparation of test solutions in the preliminary test.
Addition of test item or aniline
1) Test solution (water + test item) (n=1, Vessel No. 1)
In one test vessel, 30 mg of the test sample was accurately weighed and added to 300 mL of purified water, so that the concentration of the test item reached 100 mg/L.
2) Test solutoin (sludge + test item) (n=3, Vessel Nos 2, 3 and 4)
In each test vessel, 30 mg of the test sample was accurately weighed and added to the basal culture medium [the volume subtracting the volume (2.74 mL) of activated sludge from 300 mL], so that the concentration of the test item reached 100 mg/L.
3) Test solution (sludge + aniline) (n= 1, Vessel No. 6)
In one test vessel 29.5 µL (30 mg) of aniline was taken out by microsyringe and added to the basal culture medium [the volume subtracting the volume (2.74 mL) of activated sludge from 300 mL], so that the concentration of aniline reached 100 mg/L.
4) Test solution (control blank) (n=1, Vessel No. 5)
In one test vessel, nothin was added to the basal culture medium [the volume subtracting the volume (2.74 mL) of activated sludge from 300 mL].
Innoculation of activated sludge
The activated sludge was added to each test vessel described above so that the concentration of the suspended solid reached 30 mg/L.

TEST SYSTEM
Instruments and conditions for incubation
a) Instruments for incubation
Closed system oxygen consumption measuring apparatus
Temperature controlled bath with measuring unit
OM3001A (Ohkura Electric)
Data sampler OM7000A (Ohkura Electric)
Vessel Glass vessel
Absorbant for carbon dioxide
Soda lime No. 1
b) Conditions for incubation
Incubation temperature 25 +/- 1°C
Incubation duration 28 days (under dark conditions)
Stirring method Each test solution was stirred by a stirrer

SAMPLING
Observation and measurement
a) Observation of test solution
During the incubation period, BOD of the test solutions was measured continuously by a closed system oxygen consumption measuring appaaratus. The incubation temperature was measured and recorded once a day.
Analysis of test solution
After the end of the incubation, dissolved organic carbon (DOC) and the test item in the test solutions was determined.

Reference substance

Reference substance:

aniline

Results and discussion

% Degradation

% Degradation 1

Parameter:

other: % degradation (BOD)

Value:

98

Sampling time:

28 d

% Degradation 2

Parameter:

% degradation (DOC removal)

Value:

96

Sampling time:

28 d

% Degradation 3

Parameter:

% degradation (test mat. analysis)

Value:

100

Sampling time:

28 d

Any other information on results incl. tables

Appearances of test solutions

Appearances of test media in incubation vessels were as follows:

	Test Solution	Appearance (visual)	pH
At the start of incubation	Water + test item	The test item was not dissolved The test solution was colorless	-
	Sludge + test item	The test item was not dissolved The test solution was colorless	-
	Control blank	Insoluble compound except the sludge was not observed The test solution was colorless	-
At the end of incubation	Water + test item	Insoluble compound was not observed The test solution was colorless	Vessel No. 1 6.0
	Sludge + test item	Insoluble compound except the sludge was not observed Growth of the sludge was observed (Vessel No. 4) Growth of the sludge was not observed (Vessels Nos. 2, 3) The test solution was colorless	Vessel No. 2 7.2 Vessel No. 3 7.2 Vessel No. 4 7.2
	Control blank	Insoluble compound except the sludge was not observed The test solution was colorless	Vessel No. 5 7.4

Analytical results of test solutions

Anlaytical results of the test solutions after 28 days were as follows. The results of TOC sample-1 were rejected and those of TOC sample-2 were adopted.

		Water + test item	Sludge + test item			Theoretical amount
		Vessel No. 1	Vessel No. 2	Vessel No. 3	Vessel No. 4
BOD*^3	mg	0.3	59.8	61.5	56.1	60.0
Residual amount and percentage residue of DOC*^3(TOC sample-1, rejected)	mgC	23.2	0.6	0.7	1.2	19.3
	%	120	3	4	6	-
Residual amount and percentage residue of DOC (TOC sample-2, adopted)	mgC	20.5	0.8	0.8	1.2	19.3
	%	106	4	4	6	-
Residual amount and percentage residue of test item (by HPLC)	mg	30.1	0	0	0.5	30.0
	%	100	0	0	2	-
Produced amount and percentage production of converted product*^4(by HPLC)	mg	0	0	0	0.6	30.0
	%	0	0	0	2	-

*³ The value of the test solution (control blank) was subtracted from the values of the test solutions (sludge + test item)

*⁴ Pne converted product was detected in the test vessel No. 4 and the produced amount was calculated from the peak area of the converted product for that of the standard solution of the test item.

Percentage Biodegradation

Percentage biodegradation after 28 days were as follows. the percentage biodegradation by DOC was calculated from the analytical value obtained in TOC sample-2.

		Sludge + test item
		Vessel No. 2	Vessel No. 3	Vessel No. 4	Average
Percentage biodegradation by BOD	%	100	102	93	98
Percentage biodegradation by DOC	%	96	96	94	96
Percentage biodegradation of test item (by HPLC)	%	100	100	98	99

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The percentage biodegradation of the test item by BOD, DOC and HPLC was 98%, 96% and 99% respectively. These results indicate that the test item underwent biodegradation under the test conditions. However, the percentage residue of the test item was 2%, and the percentage production of one converted product was 2% in only one of the three test solutions (sludge + test item). BOD curve of the test solution containing them shows an upward tendency at the end of the incubation and no peaks of the test item and the converted product were detected in the other test solutions. Therefore, they will undergo biodegradation and disappear eventually. Although identification of the converted product was not performed, the converted product may be 3-ethoxy-4 -hydroxybenzaldehyde, which is readily biodegradable, because the retention time of the converted product was longer than that of the test item on the HPLC Chromatogram, and the converted product had ultraviolet absorption.

Executive summary:

The study was aimed at evaluating the biodegradability of NACET00470 by microorganisms according to OECD Guideline 301C.

Conditions of incubation:

Concentration of test item:              100 mg/L

Concentration of activated sludge    30 mg/L (as concentration of suspended solid)

Volume of test solution                   300 mL

Incubation temperature                   25 +/- 1°C

Incubation duration                          28 days (under dark conditions)

Measurement and analysis for calculation of percentage biodegradation

a)       Measurement of biochemical oxygen demand (BOD) with a closed system oxygen consumption measuring apparatus

b)       Determination of dissolved organic carbon (DOC) by a total organic carbon analysis (TOC)

c)       Determination of test item by high-performance liquid chromatography (HPLC)

Results

		Sludge + test item
		Vessel No. 2	Vessel No. 3	Vessel No. 4	Average
Percentage biodegradation by BOD	%	100	102	93	98
Percentage biodegradation by DOC	%	96	96	94	96
Percentage biodegradation of test item (by HPLC)	%	100	100	98	99

Conclusion

The percentage biodegradation of the test item by BOD, DOC and HPLC was 98%, 96% and 99% respectively. These results indicate that the test item underwent biodegradation under the test conditions. However, the percentage residue of the test item was 2%, and the percentage production of one converted product was 2% in only one of the three test solutions (sludge + test item). BOD curve of the test solution containing them shows an upward tendency at the end of the incubation and no peaks of the test item and the converted product were detected in the other test solutions. Therefore, they will undergo biodegradation and disappear eventually. Although identification of the converted product was not performed, the converted product may be 3-ethoxy-4 -hydroxybenzaldehyde, which is readily biodegradable, because the retention time of the converted product was longer than that of the test item on the HPLC Chromatogram, and the converted product had ultraviolet absorption.