Cyperus scariosus, ext.

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07.02.2018-18.05.2018

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 202 (Daphnia sp. Acute Immobilisation 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 : Caprilo (Cyperus Scariosus ext. Oil)

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

Physical appearance : Amber coloured or Light Brown Liquid

Purity as per certificate of analysis : 100% Naturally pure

Batch No. : Lot # 11/CYP/2016

Manufactured date : November 2016

Expiry 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

Sampling and analysis

Analytical monitoring:

not specified

Details on sampling:

not specififed

Test solutions

Vehicle:

no

Details on test solutions:

The medium used for the test was reconstituted water, as described in the OECD test guideline 202 (OECD, 2004). The medium was prepared as per the details given in Appendix 2.

All chemicals used in the study were of either analytical reagent (AR) or better grade reagent.

Chemical Batch No. Manufacturer
Magnesium sulphate. 7H2O H14A/1813/1707/31 s. d. fine-chem Limited
Mumbai-400030
India

E.D.T.A-disodium salt F10Z/1710/1705/31
Methyl orange indicator D16Z/1112/2402/74
Ammonium chloride D14A/2314/2103/31
Patton & Meders indicator F11Z/0311/1102/31
Ammonium hydroxide L11A/0711/2202/31
Calcium chloride, 2H2O D14A/1314/0804/31
Sodium hydroxide D16A/0415/2112/31
Hydroxyl ammonium chloride 1504/17101-1 Qualigens Fine Chemicals
Navi Mumbai – 400 710
India
Potassium chloride QJ3P631025 Merck Specialities Private Limited
Mumbai-400018
India
Sodium carbonate QC2Q620547
Erichrome Black T AL0A601067
Sodium chloride QL4Q642763
Phenolphthalein Indicator 0000245703 Himedia laboratories Pvt. Ltd.
Mumbai – 400086, India
Sodium bicarbonate 0000230345
Hydrochloric acid SHBG4416V Sigma Aldrich, 3050 Spruce street, St. Louis, USA

Test item stock of 1 mg/mL was prepared by mixing 100 mg of test item in test medium and making up the final volume to 100 mL using test medium.

To prepare test concentrations of 0.4 (G2), 0.88 (G3), 1.94 (G4), 4.27 (G5), 9.39 (G6), 20.66 (G7) and 45.46 (G8) mg/L, 0.4, 0.88, 1.94, 4.27, 9.39, 20.66, and 45.46 mL of test item stock was added and volume was made up to 1 L in a volumetric flask using test medium separately.

To prepare test concentrations of 100 mg/L, 100 mg of test item was mixed and made up to 1 L in a volumetric flask using test medium.

1 L of test medium will be used as negative control (G1).

A stock solution of reference substance (Potassium dichromate- K2Cr2O7) of 1 mg/mL was prepared by mixing 10 mg in 10 mL of test medium.

G10 (Reference substance 0.32 mg/L): 0.32 mL of stock of reference substance was mixed and made up to 1 L in a volumetric flask using test medium.

150 mL each of negative control, test item concentrations and reference substance was dispensed separately into 4 test containers.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna was used as test system as it is a recommended species in the guideline.
6.1.2.1 Source
Ecotoxicology
Eurofins Advinus Limited
Bengaluru
India
6.1.2.2 Maintenance of Daphnia
Daphnia were maintained in the test medium. The test medium in which the daphnia were maintained was changed at least twice a week and they were fed with the unicellular green alga, Chlorella vulgaris.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

not specififed

Test temperature:

20 ± 1 oC

pH:

ca. 7.8

Dissolved oxygen:

not specififed

Salinity:

not specififed

Conductivity:

not specififed

Nominal and measured concentrations:

0.88, 1.94, 4.27, 9.39, 20.66, 45.46 and 100 mg/L

Details on test conditions:

6.2.1 Outline of the Method
Neonates of daphnia aged less than 24 hour old were exposed to a series of concentrations of the test item, Caprilo (Cyperus Scariosus ext. Oil) for 48 hours during range finding test and definitive test. Certain concentrations result in certain percentage of daphnia no longer being capable of swimming at 48 hours. The effects on the organisms were assessed at 24 and 48 hours interval and the EC50 value was calculated.
6.2.2 Neonates of Daphnia
One day prior to the start of the treatment, about 100 daphnia during range finding test and definitive test were incubated in approximately 2 L of test medium. The following day, the young ones hatched which were aged less than 24 hour old were collected and used in the test.
6.2.3 Exposure to Treatment
Daphnia were exposed to the treatment under the following conditions during range finding and the definitive test.

Parameters Range finding test Definitive test
Method of exposure Static Static
Duration of the test 48 ± 1 hours 48 ± 1 hours
Test containers 250 mL glass beakers 250 mL glass beakers
No. of daphnia Ten daphnia per group divided into two replicates of five daphnia each Twenty daphnia per group divided into four replicates of five daphnia each
Loading About 15 mL of test medium to each daphnia About 30 mL of test medium to each daphnia
Light Complete darkness Complete darkness
Temperature of growth chamber (C) 20.4 to 20.5 20.3 to 20.5
Dissolved oxygen concentration of test concentration (mg/L) 7.2 to 7.4 7.2 to 7.4
pH of test concentration 7.73 to 7.83 7.73 to 7.83
Aeration The test containers were not bubble aerated The test containers were not bubble aerated
Feeding None None
6.2.4 Test Item Solubility
One gram (100 mg) of the test item was mixed and made up to 10 mL using test medium in a volumetric flask.
6.2.4.1 Range Finding Test
Neonates of daphnia were tested for immobility in test concentrations of Caprilo (Cyperus Scariosus ext. Oil) at 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 mg/L along with a negative control.
6.2.5 Definitive Test
Based on the results of the range finding test, Caprilo (Cyperus Scariosus ext. Oil) was tested at the concentrations of 0.4, 0.88, 1.94, 4.27, 9.39, 20.66, 45.46 and 100 mg/L (factor of 2.2) were tested in the definitive test. Neonates of daphnia were tested for immobility in these test concentrations along with a negative control and a reference substance.
6.2.5.1 Preparation of Test Concentrations
Test item stock of 1 mg/mL was prepared by mixing 100 mg of test item in test medium and making up the final volume to 100 mL using test medium.

To prepare test concentrations of 0.4 (G2), 0.88 (G3), 1.94 (G4), 4.27 (G5), 9.39 (G6), 20.66 (G7) and 45.46 (G8) mg/L, 0.4, 0.88, 1.94, 4.27, 9.39, 20.66, and 45.46 mL of test item stock was added and volume was made up to 1 L in a volumetric flask using test medium separately.

To prepare test concentrations of 100 mg/L, 100 mg of test item was mixed and made up to 1 L in a volumetric flask using test medium.

1 L of test medium will be used as negative control (G1).

A stock solution of reference substance (Potassium dichromate- K2Cr2O7) of 1 mg/mL was prepared by mixing 10 mg in 10 mL of test medium.

G10 (Reference substance 0.32 mg/L): 0.32 mL of stock of reference substance was mixed and made up to 1 L in a volumetric flask using test medium.

150 mL each of negative control, test item concentrations and reference substance was dispensed separately into 4 test containers.
6.2.6 Observations
Following parameters were observed and recorded during both range finding test and definitive test.

pH and dissolved oxygen concentration of test concentrations at the start and end of the treatment period were recorded.

Immobility of daphnia at 24 ± 1 and 48 ± 1 hour after the start of the treatment was recorded.

The temperature of the incubator/growth chamber was recorded once daily.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 5.655 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The EC50 value for Caprilo (Cyperus Scariosus ext. Oil) at 48 hours was 5.655 mg/L based on nominal concentrations with fiducial limits at 95 % being 3.8905 and 8.2185 mg/L.

Executive summary:

1.1.1                      Outline of the Method

Neonates of daphnia aged less than 24 hour old were exposed to a series of concentrations of the test item,Caprilo (Cyperus Scariosus ext. Oil)for 48 hours during range finding test and definitive test. Certain concentrations result in certain percentage of daphniano longer being capable of swimming at 48 hours. The effects on the organisms were assessed at 24 and 48 hours interval and the EC₅₀value was calculated.

1.1.2                      NeonatesofDaphnia

One day prior to the start of the treatment, about 100 daphnia during range finding test and definitive test were incubated in approximately 2 L of test medium. The following day, the young ones hatched which were aged less than 24 hour old were collected and used in the test.

1.1.3                      Exposure to Treatment

Daphnia were exposed to the treatment under the following conditions during range finding and the definitive test.

 

Parameters	Range finding test	Definitive test
Method of exposure	Static	Static
Duration of the test	48 ± 1 hours	48 ± 1 hours
Test containers	250 mL glass beakers	250 mL glass beakers
No. of daphnia	Ten daphniaper group divided into two replicates of five daphnia each	Twenty daphniaper group divided into four replicates of five daphnia each
Loading	About 15 mL of test medium to each daphnia	About 30 mL of test medium to each daphnia
Light	Complete darkness	Complete darkness
Temperature of growth chamber (°C)	20.4 to 20.5	20.3 to 20.5
Dissolved oxygen concentration of test concentration (mg/L)	7.2 to 7.4	7.2 to 7.4
pH of test concentration	7.73 to 7.83	7.73 to 7.83
Aeration	The test containers were not bubble aerated	The test containers were not bubble aerated
Feeding	None	None

1.1.4                      Test Item Solubility

One gram (100 mg) of the test item was mixed and made up to 10 mL using test medium in a volumetric flask.

1.1.4.1                Range Finding Test

Neonates of daphnia were tested for immobility in test concentrations of Caprilo (Cyperus Scariosus ext. Oil) at 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 mg/L along with a negative control.

1.1.5                      Definitive Test

Based on the results of the range finding test, Caprilo (Cyperus Scariosus ext. Oil) was tested at the concentrations of 0.4, 0.88, 1.94, 4.27, 9.39, 20.66, 45.46 and 100 mg/L (factor of 2.2) were tested in the definitive test. Neonates of daphnia were tested for immobility in these test concentrations along with a negative control and a reference substance.

1.1.5.1                Preparation ofTestConcentrations

Test item stock of 1 mg/mL was prepared by mixing 100 mg of test item in test medium and making up the final volume to 100 mL using test medium.

 

To prepare test concentrations of 0.4 (G2), 0.88 (G3), 1.94 (G4), 4.27 (G5), 9.39 (G6), 20.66 (G7) and 45.46 (G8) mg/L, 0.4, 0.88, 1.94, 4.27, 9.39, 20.66, and 45.46 mL of test item stock was added and volume was made up to 1 L in a volumetric flask using test medium separately.

 

To prepare test concentrations of 100 mg/L, 100 mg of test item was mixed and made up to 1 L in a volumetric flask using test medium.

 

1 L of test medium will be used as negative control (G1).

 

A stock solution of reference substance (Potassium dichromate- K₂Cr₂O₇) of 1 mg/mL was prepared by mixing 10 mg in 10 mL of test medium.

 

G10 (Reference substance 0.32 mg/L): 0.32 mL of stock of reference substance was mixed and made up to 1 L in a volumetric flask using test medium.

 

150 mL each of negative control, test item concentrations and reference substance was dispensed separately into 4 test containers.

1.1.6                      Observations

Following parameters were observed and recorded during both range finding test and definitive test.

 

pH and dissolved oxygen concentration of test concentrations at the start and end of the treatment period were recorded.

 

Immobility of daphnia at 24 ± 1 and 48 ± 1 hour after the start of the treatment was recorded.

 

The temperature of the incubator/growth chamber was recorded once daily.