Cyperus scariosus, ext.

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:

01.03.2018-28.06.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Cyperus scariosus, ext.

EC Number:

294-955-2

EC Name:

Cyperus scariosus, ext.

Cas Number:

91771-62-9

IUPAC Name:

Cyperus scariosus, ext.

Test material form:

liquid

Specific details on test material used for the study:

Batch manufactured and supplied by : Jagat Aroma Oils Distillery
Kannauj-209725
Uttar Pradesh, India

Test item : Cypriol (Cyperus Scariosus ext. Oil)

CAS No. : 91771-62-9/68916-60-9

Physical appearance : Amber coloured or Light Brown Liquid Liquid

Purity as per certificate of analysis : 100% Naturally pure

Total Organic Content (%) : 81.62

Lot No. : # 11/CYP/2016

Manufactured date : November 2016

Retest date : October 2018

Recommended storage condition : Ambient (+15 to +25 °C)


Notes: a) Date of receipt of test item at test facility: 05 December 2017
b) Test Item code by test facility: J09-01

Study design

Oxygen conditions:

aerobic/anaerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

The inoculum was secondary effluent, collected from a treatment plant receiving predominantly domestic sewage. This effluent was used as test system as it is recommended in the guideline. A fresh sample of secondary effluent was collected from the treatment plant and was kept aerobic during transport.

This effluent was allowed to settle for one hour, decanted and the decanted effluent was used in the test.

Source of the Inoculum

Sewage Treatment Plant
Eurofins Advinus Limited
Bengaluru – 560 058
India

The bacterial population in the inoculum was determined as colony forming units (CFU/mL) by diluting the inoculum to an appropriate dilution and then plating on nutrient agar plates.

The decanted effluent was preconditioned by aerating for 5 days at 22 to 24C.

The bacterial population in the inoculum was 6.0 x 107 CFU/L

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

ca. 190 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

6.1.3 Reference Item
Analytical grade Sodium benzoate was used as the reference item.

Batch No.: A117661401
Manufactured By: Loba Chemie Pvt. Ltd., Mumbai, India
6.1.4 Test Medium and Chemicals
Analytical or better grade chemicals were used in the study.

Chemical Batch No. Manufactured by
Nutrient agar 0000230748 Himedia Laboratories Pvt. Ltd., Mumbai, India

Phenolphthalein indicator C20H14O4 0000245703
Dipotassium hydrogen orthophosphate, K2HPO4 DB7D670111 Merck Specialities Pvt. Ltd., Mumbai, India
Methyl Orange pH indicator D162/1112/2402/74 SD fine chem. Ltd.
Mumbai, India

Iron (III) chloride hexahydrate, FeCl3.6H2O G173/1216/1403/02
Potassium dihydrogen orthophosphate, KH2PO4 C16A/0715/2412/31
Sodium carbonate, Na2CO3 B16A/0916/0102/31
Calcium chloride, dihydrate, CaCl2.2H2O D14A/1314/0804/31
Ammonium chloride, NH4Cl D14A/2314/2103/31
Disodium hydrogen orthophosphate dihydrate, Na2HPO4.2H2O D14A/0513/0608/31
Magnesium sulphate heptahydrate, MgSO4.7H2O H14A/1813/1707/31
Potassium hydrogen phthalate,
COOH.C6H4.COOK 2184160314 Thermofisher Scientific India Pvt. Ltd., Mumbai, India
Barium hydroxide Ba(OH)2 15505 Thomas Baker (chemicals) Pvt Ltd, Mumbai, India
Hydrochloric acid, HCl SHBG4416V Sigma Aldrich,
St.Louis, USA
6.1.5 Equipment
a) System for gas flow control
b) Bubble flow meter
c) Conical flasks – 5 L capacity each fitted with an aeration tube reaching nearly to the bottom of the flask and an outlet
d) Gas absorption bottles
e) Glass tube, rubber corks with two holes
f) Zero air cylinder
g) Apparatus for carbon dioxide scrubbing
h) Burette, pipettes and other laboratory apparatus
i) Balance: Model: PB 303 and AG 245, Mettler Toledo, Switzerland
j) pH meter: Model: Eutech pH/mv/ºC meter, Eutech Instruments, Singapore
k) Milli-Q unit: Model: Milli-Q, Millipore India Private Limited, Bengaluru, India
6.2 Methods
6.2.1 Outline of the Method
A measured volume of the inoculated mineral medium, containing a known concentration of the test item [10-20 mg total organic carbon (TOC) per litre] as the nominal sole source of organic carbon was aerated by the passage of carbon dioxide-free air at a controlled rate in the dark. Degradation was followed over 28 days by determining the carbon dioxide produced. The carbon dioxide was trapped in barium hydroxide and was measured by titration of the residual hydroxide or as inorganic carbon. The amount of carbon dioxide produced from the test item (corrected for that derived from the blank inoculum) was expressed as a percentage of theoretical carbon dioxide (ThCO2).
6.2.2 Test Item Solubility
The test item was tested for its solubility in mineral media at 240 mg/L.
6.2.3 Preparation of Test Medium
The stock solutions and the test medium were prepared as per the compositions of chemicals given in Appendix 2. High quality ultra-pure water delivered by a Milli-Q system was used to prepare the stock solutions and the mineral medium.
6.2.4 Performance of the Test
Test System Identification

Test flasks were labeled to indicate the study number and the flask numbers.

Preparation of Test Flasks

Flask No. Contents
1 & 2 Test suspension – test item and inoculum
3 & 4 Inoculum blank – only inoculum
5 Procedure control – reference item and inoculum
6 Toxicity control – test item, reference item and inoculum

To each 5 L flask, 2400 mL of mineral medium was added and mixed with 300 mL of the pre-conditioned inoculum. A separate 3000 mL of mineral medium was also prepared in a flask to use it for further dilutions.

A sample of the mineral medium was checked for the inorganic carbon content.

These flasks were aerated with CO2 free air at 43 to 50 mL/minute, overnight to purge the system of carbon dioxide.

Inoculum Blanks

To the test flasks numbered 3 and 4 (inoculum blanks), 300 mL each of mineral medium (previously aerated with CO2-free air) was added to make the total suspension volume to 3000 mL in each flask.




Addition of Test Item and Reference Item

The total organic carbon (TOC) in the test as well as the reference item was determined using the formula:


% TOC
= Carbon content of the test/reference item
x
100
Molecular weight of the test/reference item

% TOC of Test item – 81.62
Molecular formula of reference item (Sodium benzoate) – C7H5NaO2

Based on the TOC, 57 mg of test item was weighed and made up to 300 mL using mineral medium (previously aerated with CO2-free air) and added to each test flasks 1 and 2 separately. Similarly, 78 mg of reference item was made up to 300 mL with mineral medium (previously aerated with CO2-free air) and added to test flask 5. A quantity of 29 mg test item and 39 mg of the reference item was mixed and made up to 300 mL using mineral medium (previously aerated with CO2-free air) and added to test flask 6.

Exposure to Treatment

The outlet of each test flask was connected to the inlet of a gas absorption bottle containing 100 mL of 0.0125 M barium hydroxide solution in a series of 3 keeping the outlet of the last absorption bottle open.

Before each use, the strength of barium hydroxide was determined by titrating against potassium hydrogen phthalate (PHP). For this, 10 mL of 0.025 M PHP was transferred into a conical flask, 2 to 3 drops of phenolpthalein indicator was added and this was titrated against 0.0125 M barium hydroxide solution until the colour of PHP just turned pink. The molarity of barium hydroxide was calculated using the formula –

Molarity of Barium hydroxide = Volume of PHP taken x Molarity of PHP
2 x Titre value

The test was carried out by bubbling carbon dioxide free air through the suspension at a rate of 43 to 50 mL/minute and continued for 28 days. Once a week, the flow rate of carbon dioxide free air was checked using the bubble flow meter.

On the 28th day, pH of the test suspension was recorded and 1 mL of concentrated hydrochloric acid was added to each flask and the bubbling of carbon dioxide free air was continued.

On the 29th day, the bubbling was stopped. All the barium hydroxide absorption bottles were disconnected and were titrated for the determination of carbon dioxide production.

The temperature of the room was maintained at 22 to 24ºC during the treatment period.
6.2.5 Observations
6.2.5.1 Determination of CO2
To identify the 10-d window period, during the first 10 days, carbon dioxide analysis was made every second-third day and then at least every fourth day until the 29th day.
6.2.5.2 Data Presentation
The amount of CO2 produced was calculated from the amount of base remaining in the absorption bottle. The amount of CO2 remaining is assessed by titrating 0.0125 M Ba(OH)2 with 0.05 M HCl [Thus 50 ml HCl would be needed to titrate 100 ml Ba(OH)2].

Since, 1 m mol of CO2 is produced for every m mol of Ba(OH)2 reacted to BaCl2 and 2 m mol of HCl are needed for the titration of the remaining Ba(OH)2 and given that the molecular weight of CO2 is 44 g, the weight of CO2 produced (mg) was calculated by:

CO2 produced (mg) = Molarity of HCl x (50 – mL HCl titrated) x 44
2

The weights of CO2 produced from the inoculum alone and from the inoculum plus test item was calculated using the respective titration values; the difference is the weight of CO2 produced from the test item alone.

The percentage biodegradation is calculated from:

% degradation = mg CO2 produced x 100
mg TOC added in test x 3.67

where 3.67 was the conversion factor (44/12) for carbon to carbon dioxide.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

% Degradation 1

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 12.79

St. dev.:

1.92

Sampling time:

10 d

% Degradation 2

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 34.57

St. dev.:

3.33

Sampling time:

28 d

BOD5 / COD results

Results with reference substance:

118.16

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Based on the results, it was concluded that the test item, Cypriol (Cyperus Scariosus ext. Oil) is biodegradable but not readily biodegradable as 34.57% mean degradation was achieved at the end of test which is less than 60% pass level in 10-day window period within the 28-d period of the test.

Executive summary:

The ready biodegradability of Cypriol (Cyperus Scariosus ext. Oil) was tested using the CO₂Evolution Test. The test item was added to two test vessels at the concentration of 19 mg/L (equivalent to 15.5 mg of Total Organic Carbon/L). Two control treatments containing only the inoculum, one reference item treatment and one toxicity control treatment containing the test item and the reference item were also tested. All the treatments were added with equal volume of inoculum which was collected from the secondary effluent treatment plant receiving predominantly domestic sewage.

 

Treatment mixtures were aerated for 29 days with carbon dioxide (CO₂) free air. The CO₂released was trapped in a series of bottles containing barium hydroxide, which were connected to the outlet of each test vessel. The residual barium hydroxide was measured on Days 3, 6, 8, 10, 13, 16, 20, 24, 27 and 29 after the initiation of the test.

 

The mean per cent degradation of test item was 34.57% at the end of test while, the percent degradation of reference item was 118.16% and the toxicity control was 82.68% at the end of the test. It was observed that the mean degradation of test item, Cypriol (Cyperus Scariosus ext. Oil) was 34.57% which is less than 60% pass level in 10-day window period within the 28-d period of the test.

 

The test fulfilled all the validity criteria.

 

Based on the results, it was concluded that the test item, Cypriol (Cyperus Scariosus ext. Oil) is biodegradable but not readily biodegradable as 34.57% mean degradation was achieved at the end of test which is less than 60% pass level in 10-day window period within the 28-d period of the test.