Apple, Malus sylvestris, 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:

November - December 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:

2018

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:

Apple, Malus sylvestris, ext.

EC Number:

286-475-7

EC Name:

Apple, Malus sylvestris, ext.

Cas Number:

85251-63-4

IUPAC Name:

Apple, Malus sylvestris, ext.

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

According to the results of the preliminary test, we proposed to test five concentrations for this study: 10 – 32 – 102 – 328 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 algal 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 algal growth inhibition in relation to the control was evaluated over a period of 72 hours. The algal 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.

The 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.
The final test contained:
 Control: 6 biotic replicates + 1 abiotic replicate
 Concentration 10 mg/L: 3 biotic replicates + 1 abiotic replicate
 Concentration 32 mg/L: 3 biotic replicates
 Concentration 102 mg/l: 3 biotic replicates
 Concentration 328 mg/L: 3 biotic replicates + 1 abiotic replicate
 Concentration 1050 mg/L: 3 biotic replicates + 1 abiotic replicate

The replicates were pooled before chemical analyses.
The abiotic replicates were used for chemical analyses only

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 - 328 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:

22.4 °C – 23.0°C

pH:

8.0 (Algae test medium)

Dissolved oxygen:

no data

Salinity:

no data

Conductivity:

no data

Nominal and measured concentrations:

Control - 10- 32 – 102 – 328 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, 328 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 :6163 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 – 328 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 cocentration

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

yield

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 cocentration

Details on results:

The results obtained for the test item on the toxicological endpoint “inhibition of growth rate” indicate an ErC10-(0-72h) higher than 1050 mg/L(highest tested concentration) value expressed in nominal test item concentration.
The results obtained for the test item on the toxicological endpoint “inhibition of growth rate” indicate an ErC50-(0-72h) higher than 1050 mg/L (highest tested concentration) value expressed in nominal test item concentration.

The results obtained for the test item on the toxicological endpoint “Yield inhibition” indicate an EyC10-(0-72h) higher than 1050 mg/L(highest tested concentration) value expressed in nominal test item concentration.

The results obtained for the test item on the toxicological endpoint “Yield inhibition” indicate an EyC50-(0-72h) higher than 1050 mg/L (highest tested concentration) 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 algal growth inhibition test performed with the reference item K2Cr2O7 (September 18, 2017) gave an ErC50(0-72h) = 1.11 mg/L (0.98 – 1.24), indicating that the algal 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 on the toxicological endpoint “inhibition of growth rate” indicate an ErC50-(0-72h) higher than 1050 mg/L (highest tested concentration) 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 on the growth of the unicellular green alga Pseudokirchneriella subcapitata, according to the OECD 201 guideline (March 2006, corrected July 2011).

The results obtained for the test item on the toxicological endpoint “inhibition of growth rate” indicate an ErC₅₀-(0-72h)higher than 1050 mg/L (highest tested concentration) 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 successfully fulfilled.