Magnesium octadecylbenzenesulphonate

Biodegradation in water: screening tests

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Administrative data

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept 2016 to Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

solid: crystalline

Details on test material:

Batch No.: E00873-025-001
Physical Description: Black solid
Storage Conditions: Kept in a controlled room temperature area

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

On-site sludge sampling was carried out at 10 locations in Japan (samples were from surface water and surface soil of rivers, lake and inland sea, and return sludge from sewage plants). Activated sludge, which was prepared and was controlled in this laboratory according to the test method described in Section 5., was used in this study (sampling period: July, 2016, initiation date of use: August 19, 2016).

The activated sludge, which was cultivated for 18.5 hours after feeding with the synthetic sewage, was used. The synthetic sewage 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

Initial test substance concentration

Initial conc.:

30 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation 1

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation 2

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

Preparations for test
a. Decision of additive amount of activated sludge
Additive amount of activated sludge into the test vessels was 8.67 mL. That into the test vessel was 2.60 mL. The additive amounts were based on 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 0102:2016 Section 14.1 Date September 12, 2016
Result 3460 mg/L
b. Preparation of basal culture medium
The basal culture medium (5 L) was prepared at the same proportion as the following method; purified water (The Japanese Pharmacopoeia, Takasugi Pharmaceutical) was added to each 3 mL aliquot of solutions A, B, C and D, which are described in JIS K 0102:2016 Section 21, in order to prepare 1 L of solution. The pH of this solution was adjusted to 7.0.
c. Validity of activated sludge
The activated sludge was confirmed to be sufficiently active by using aniline as a reference item.

The following test solutions were prepared and incubated under the conditions described in Section 13.3. The pH of the test solutions was not measured at the preparation of them because the test item was not dissolved in the test solution
a) Addition of test item or aniline
• Test solution (water + test item) (n=1, Vessel No. 1)
In one test vessel, 10 mg of the test sample (9 mg as the test item) was accurately weighed with an electronic analytical balance and added to 300 mL of purified water, so that the concentration of the test item reached 30 mg/L.
• Test solution (sludge + test item) (n=3, Vessel Nos. 2, 3 and 4)
In each test vessel, 10 mg of the test sample (9 mg as the test item) was accurately weighed with an electronic analytical balance and added to the basal culture medium [the volume subtracting the volume (8.67 mL) of activated sludge from 300mL1, so that the concentration of the test item reached 30 mg/L.
• Test solution (sludge + aniline) (n=1, Vessel No. 6)
In one test vessel, 29.5 µL (30 mg) of aniline was taken out with micro-syringe and added to the basal culture medium [the volume subtracting the volume (2.60 mL) of activated sludge from 300 mL], so that the concentration of aniline reached 100 mg/L.
• Test solution (control blank) (n=1, Vessel No. 5)
In one test vessel, nothing was added to the basal culture medium [the volume subtracting the volume (8.67 mL) of activated sludge from 300 mL.
b) Inoculation of activated sludge
• The activated sludge was added to each test vessel described in 2) and 4), so that the concentration of the suspended solid reached 100 mg/L. That was added to the test vessel described in 3) to reach 30 mg/L in the concentration of the suspended solid.
I
nstruments and conditions of incubation
a. Instruments for incubation
• Closed system oxygen consumption measuring apparatus
• Temperature controlled bath with measuring unit
• Absorbent for carbon dioxide
o Soda lime No.1 (for absorption of carbon dioxide, Wako Pure Chemical Industries)
b. Conditions of incubation
• Incubation temperature - 25±1°C
• Incubation duration - 28 days (under dark conditions)
• Stirring method - Each test solution was stirred with a stirrer.

Observation and measurement
a. Observation of test solution
• During the incubation period, the appearance of the test solution was observed once a day.
a. Measurement of biochemical oxygen demand (BOD)
• During the incubation period, 130D of the test solutions was measured continuously with a closed system oxygen consumption measuring apparatus. The incubation temperature was measured and recorded once a day

After the end of the incubation, the test item in the test solution was determined. In addition, the pH of the test solutions was measured.

The test solution (water + test item), the test solutions (sludge + test item) and the test solution (control blank) were pretreated to prepare samples for high-performance liquid chromatography (HPLC) analysis.

The test material was isolated using a chloroform solvent extraction technique, it was then dried on filter papaer and subsequently dissolved in THF, before being analyzed quantitatively by HPLC.

The test item was determined with absolute calibration curve method using one concentration of standard solution.

In order to confirm the validity of this determination method, the calibration curve was made using four concentrations of standard solutions, 18.0, 451, 902 and 1800 mg/L (see Fig. 2). As a result, the regression line of the calibration curve was a straight line from the origin, therefore the validity was confirmed.

Analytical conditions
Instrument High-performance liquid ehromatograph
Pump LC-20AD (Shimadzu)
UV-VIS detector SPD-20AV (Shimadzu)
Column oven CTO-20A (Shimadzu)
Auto injector SIL-20ACHT (Shimadzu)
System controller SCL-10Avp (Shimadzu)
Degasser DGU-20A3 (Shimadzu)
Column Shodex Asahipak GF310A-4E
(250 mm x 4.6 min I.D., SHOWA DENKO)
Column temperature 40°C
Eluent Tetrahydrofuran
Flow rate 0.2 ml/min
Measurement wavelength 262 urn (see Fig. 5)
Injection volume 20 pL

The test sample (168 mg, i.e., 150 mg as the test item) was accurately weighed with an electronic analytical balance and dissolved in tetrahydrofuran in order to obtain 3010 mg/L solution of the test item. The standard solution of 1800 mg/L was then prepared from this solution by dilution with tetrahydrofiran.
The concentration of the test item in the sample for HPLC analysis was calculated proportionally by comparing the peak area on the chromatogram of the sample for HPLC analysis with That on the chromatogram of 1800 mg/L standard solution (see Table-3 and Fig. 4).
Limit of quantitation of the test item was 18.0 mg/L, which was the lowest concentration of the standard solution used to prepare the calibration curve.

13.5.3 Recovery test
Two test solutions (water + test item), two test solutions (sludge + test item) and a test solution (control blank) were prepared. These test solutions were pretreated in accordance with the method above, after stiffing at room temperature for about 30 minutes. Then, the samples for HPLC analysis obtained in the pretreatment were analyzed according to the analytical conditions described above and the recovery rates of the test item were calculated. The blank peak exceeding the limit of quantitation was detected around the peak of the test item on the chromatogram of the test solution (control blank). Since the blank peak is due to the pretreatment method, the recovery rate was calculated by using the value subtracting the blank peak area from the peak area of the test solution (water + test item) or the test solutions (sludge + test item).
The average in each test solution was 90% and more, and the difference between replicate values in each was within 5%. Therefore, the validity of the pretreatment applied in this study was confirmed.
The concentrations of the test item in the analysis samples were corrected using the averages.
Recovery rate in the test solutions (water + test item) 99.4%, 94.6% average 97.0%
Recovery rate in the test solutions (sludge + test item) 99.8%, 96.0% average 97.9%
Calculation of percentage biodegradation
The percentage biodegradation was calculated by the following equations and rounded off to the
whole number.
a. Percentage biodegradation by BOD
BOD - B
Percentage biodegradation (%) = ((BOD – B)/TOD) x 100
BOD : Biochemical oxygen demand in the test solution (sludge + test item) (experimental: mg)
B : Biochemical oxygen demand in the test solution (control blank) (experimental: mg)
TOD : Theoretical oxygen demand required when the test item was completely oxidized
(theoretical: mg)
TOD was calculated with the molecular formula C69H106Mg06S2
b. Percentage biodegradation of test item.
Percentage biodegradation (%) ((Sw-Ss)/Sw) x 100
Ss : Residual amount of the test item in the test solution (sludge + test item) (experimental: mg)
Sw : Residual amount of the test item in the test solution (water + test item) (experimental: mg)
Treatment of numerical values
Values were rounded off in accordance with JIS Z 8401:1999 rule B. The atomic weight of each element used in this study was based on the 4-digit atomic weighttable 2012 provided by The Chemical Society of Japan.

Reference substance

Reference substance:

aniline

Results and discussion

% Degradation

% Degradation 1

Key result

Parameter:

% degradation (test mat. analysis)

Value:

47

Sampling time:

28 d

% Degradation 2

Key result

Parameter:

% degradation (O2 consumption)

Value:

38

Sampling time:

28 d

BOD5 / COD results

Results with reference substance:

Percentage biodegradation of aniline by BOD
71% degradation after 7days
83% degradation after 14 days

Any other information on results incl. tables

Analytical results of the test solution after 28days was as follows:

	Vessel No. 1	Vessel No. 2	Vessel No. 3	Vessel No. 4	Theoretical Amount
BOD*	0.2	7.7	10.4	9.5	24.5 **
Residual amount of test item by HPLC (mg)	8.2	5.7	3.6	3.8	9.0
Residual percentage of test item by HPLC (%)	91	63	40	42	-

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

** Calculated with the molecular formula C60H106MgO6S2.

Percentage Biodegradation

	Vessel No. 2	Vessel No. 3	Vessel No. 4	Average
Percentage Biodegradation by BOD	32	43	39	38
Percentage Biodegradation of Test Item (by HPLC)	31	56	54	47

Applicant's summary and conclusion

Validity criteria fulfilled:

no

Interpretation of results:

inherently biodegradable, not fulfilling specific criteria

Conclusions:

The average percentage biodegradation by BOD and that of the test item were 38% and 47%, respectively. In addition, growth of the sludge was observed at the end of the incubation. These results suggested that the test item is inherently biodegradable..

Executive summary:

The study was conducted in accordance with OECD guideline 302C Inherent Biodegradability Modified MITI Test. The average percentage biodegradation by BOD and that of the test item were 38% and 47%, respectively. In addition, growth of the sludge was observed at the end of the incubation. These results suggested that the test item is inherently biodegradable..