Lentil, ext.

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Lentil, ext.

EC Number:

289-998-9

EC Name:

Lentil, ext.

Cas Number:

90063-40-4

IUPAC Name:

Enzymatic hydrolysis products of Lentil extract

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Yellow powder
- Storage In darkness at room temperature
- Batch: 16 273 2 1
- Manufacturing date: September 2016
- Expiry date: September 2018

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Chemical analyses of “p-REFINYL LYO” were performed in the test solutions (biotic and abiotic replicates) according to the Study 16/1252F/d.
In order to verify the nominal or initial concentrations of the test item and maintenance of the concentrations during the ecotoxicological testing, chemical analysis of “p-REFINYL LYO” in algae test solutions were performed according to the Analytical protocol FCBA chimie no.360-e: version 2 Method for the chemical analysis of Total Carbon (TC) in samples containing the test item p-REFINYL LYO from algae ecotoxicology solutions by Carbon Analyzer (described in the study 16/1252F/d) and the following protocol.

- at the beginning of the test (T = 0h)
- at the end of the test (T = 72h)
- for the Control (i.e. algae test medium filtered), the highest and the lowest concentrations and a concentration around expected EC50

Schedule analysis at initial time (T0h):
Control Abiotic 1 replicate
10 mg/L Abiotic 2 replicates
330 mg/L Abiotic 2 replicates
1050 mg/L Abiotic 2 replicates

Schedule analysis at final time (T72h):
Control Biotic 1 replicate
10 mg/L Biotic 2 replicates
330 mg/L Biotic 2 replicates
1050 mg/L Biotic 2 replicates

Control Abiotic 1 replicate
10 mg/L Abiotic 2 replicates
330 mg/L Abiotic 2 replicates
1050 mg/L Abiotic 2 replicates

The measured concentrations of the “p-REFINYL LYO” should not vary more than ± 20 % of nominal or initial measured concentration during the test.

All the solutions were kept in refrigerator or freeze in order to be analyzed if necessary or relevant. The request of analysis was carried out by e-mail with the Sponsor

Test solutions

Vehicle:

no

Details on test solutions:

The test item was soluble in the test medium. A concentrated test solution was prepared. Dilutions were then prepared in this medium for each tested concentration.
At the request of the sponsor, the test medium and the five tested concentrations solutions were filtered through a membrane filter (cellulose acetate) with a mesh size of 0.22 μm to sterilizing and all the manipulations were done under sterile conditions to prevent contamination. Five concentrations (determined from a non-GLP preliminary test) in a geometric serie, were performed plus a Control (Test medium).
Based on the results of the preliminary Test, the following test concentrations 10 – 32 – 102 - 330 and 1050 mg test item /L were proposed for the definitive test in agreement with the Sponsor.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

Freshwater unicellular green alga Pseudokirchneriella subcapitata of the chlorococcal order (formerly named Raphidocellis subcapitata) strain number CCAP 278.4 was used in this study.
The algae cultures are stored in the laboratory on a solid medium (LC/agar Petri plates) and in a liquid medium with regular sub-culturing (every week)

Study design

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test conditions

Hardness:

no data

Test temperature:

22.8 – 23.4°C

pH:

8.1 (Algae test medium)

Dissolved oxygen:

no data

Salinity:

no data

Conductivity:

no data

Nominal and measured concentrations:

Control - 10- 32 – 102 – 330 and 1050 mg

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Initial cells density: 10000 cells/mL at the start of the test
- No. of vessels per concentration (replicates): 3 biotic replicates per concentration + 1 abiotic replicate for the concentrations: 10, 330 and 1050 mg/L
- Control: 6 biotic replicates + 1 abiotic replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Algae test medium (OECD medium); Algae dilution water

OTHER TEST CONDITIONS
- Light intensity and quality :5957 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The algae growth inhibition in relation to the control was evaluated over a period of 72 h. The algae cells per unit volume were measured during the test at 24,48 and 72 h with a particle counter (Beckman Coulter Z2) and under a microscope with a Malassez counting cell.
- pH: pH of each test concentration was measured at the start and at the end of the test. The pH of the control medium had not increased by more than 1.5 units during the test.

TEST CONCENTRATIONS
According to the results of the preliminary test, we proposed to test five concentrations for this study: 10 – 32 – 102 – 330 and 1050 mg/L.
A growing exponential phase inoculum of the freshwater unicellular alga Pseudokirchneriella subcapitata was added in order to have an initial concentration of 104 cells/mL at the start of the test: 100 mL of algae cultures grown in 250 mL erlenmeyer flasks which were placed in a cooling incubator at a temperature in the range of 21 to 24°C controlled at ± 2°C and under a constant shaking fixed at 120 rpm and a continuous illumination comprised between 4 440 and 8 880 lux.
The algae growth inhibition in relation to the control was evaluated over a period of 72 hours. The algae cells per unit volume were measured during the test at 24, 48 and 72 hours with a particle counter (Beckman Coulter Z2) and/ or under microscope with Malassez counting cell.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 050 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: highest tested concentration

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

yield

Effect conc.:

ca. 66.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The results obtained for the test item “p-REFINYL LYO” on the toxicological endpoint “inhibition of growth rate” indicate an ErC50-(0-72h) superior to 1050 mg/L (highest tested cocentration) value expressed in nominal test item concentration.

The results obtained for the test item “p-REFINYL LYO” on the toxicological endpoint “Yield inhibition” indicate an EyC50-(0-72h) equal to 66.7 mg/L [lower 95%-CI: 41.5 – upper 95%-CI: 105.4], value expressed in nominal test item concentration.

In the control. the increase of the biomass during 72 hours was determined to be higher than factor of 16.
The coefficients of variation of daily section-by-section specific growth rate and of average specific growth rate were determined respectively to be less than 35% and 7% in the control.

The validity criteria were successful fulfilled.

Results with reference substance (positive control):

The last algae growth inhibition test performed with the reference item K2Cr2O7 (January 23, 2017) gave an ErC50(0-72h) = 1.18 mg/L (1.03 – 1.36), indicating that the algae sensitivity is correct (within the accepted interval of 0.65 to 1.73 mg/L fixed by the International Standard NF EN ISO 8692 from Mai 2012 as mentioned in the OECD Guideline) and in accordance with previous data obtained by the laboratory.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The results obtained for the test item “p-REFINYL LYO” on the toxicological endpoint “inhibition of growth rate” indicate an ErC50-(0-72h) superior to 1050 mg/L (highest tested cocnetration) value expressed in nominal test item concentration.

The results obtained for the test item “p-REFINYL LYO” on the toxicological endpoint “Yield inhibition” indicate an EyC50-(0-72h) equal to 66.7 mg/L, value expressed in nominal test item concentration.

In the control. the increase of the biomass during 72 hours was determined to be higher than factor of 16.
The coefficients of variation of daily section-by-section specific growth rate and of average specific growth rate were determined respectively to be less than 35% and 7% in the control.

The validity criteria were successful fulfilled.

Executive summary:

The aim of this study was to determine the effects of the test item "p-REFINYL LYO" on the growth of the unicellular green algaPseudokirchneriella subcapitata, according to the OECD 201 guideline (March 2006, corrected July 2011).

The results of this study are summarized below:

Test item                                    p-REFINYL LYO

 Test system                              Pseudokirchneriella subcapitata

 Exposure duration                   72 hours

The 72-hour EyC₅₀of the test item“p-REFINYL LYO”was determined to be66.7 mg test item/L, value expressed in nominal test item concentration   In the control, the increase of the biomass during 72 hours was determined to be higher than factor of 16. The coefficients of variation ofdaily section-by-section specific growth rateand ofaverage specific growth ratewere determined respectively to be less than 35 % and 7% in the control. The validity criteria were successful fulfilled.
  

Test concentrations                 Control - 10- 32 – 102 – 330 and 1050 mg/L

 Incubator                              Temperature 22.8 – 23.4 °C ; shaking at 120 rpm; light intensity: 5957 Lux

 Measurement of cell growth  Beckman Coulter Z2 and Malassez counting cell

 Results

The 72-hour ErC₅₀of the test item“p-REFINYL LYO”was determined to be superior to the highest tested concentration,i.e. 1050.0 mg test item/L, value expressed in nominal test item concentration

The 72-hour EyC₅₀of the test item“p-REFINYL LYO”was determined to be 66.7 mg test item/L, value expressed in nominal test item concentration.

In the control, the increase of the biomass during 72 hours was determined to be higher than factor of 16. The coefficients of variation ofdaily section-by-section specific growth rateand ofaverage specific growth ratewere determined respectively to be less than 35 % and 7% in the control. The validity criteria were successful fulfilled.