Enzymatic hydrolyis products of Candida Saitoana

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:

March - April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

but has no incidence on study results and on compliance with Good Laboratory Practices (GLP).

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Enzymatic hydrolyis products of Candida Saitoana

EC Number:

952-608-9

IUPAC Name:

Enzymatic hydrolyis products of Candida Saitoana

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Chemical analyses of the test item were performed in the test solutions (biotic and abiotic replicates). 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 the test item in algal test solutions were performed according to the Analytical protocol FCBA chimie no.429-e: version 2, Method for the chemical analysis of Total Carbon (TC) in samples containing the test item from algae ecotoxicology solutions by a Total Organic Carbon analyzer (described in the study 18/1136F/a) 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. algal test medium filtered), the highest and the lowest concentrations and a concentration around expected EC50.

Schedule analysis at initial time (T0h):
Control Abiotic
10 mg/L Abiotic
328 mg/L Abiotic
1050 mg/L Abiotic

Schedule analysis at final time (T72h):

Control Biotic Control Abiotic
10 mg/L Biotic 10 mg/L Abiotic
328 mg/L Biotic 328 mg/L Abiotic
1050 mg/L Biotic 1050 mg/L Abiotic

The measured concentrations of the test item did 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. No request of analysis was carried out.

Test solutions

Vehicle:

no

Details on test solutions:

According to the Sponsor, the test item was soluble in the test medium.
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 0.2 µm to sterilizing and all the manipulations were performed under sterile conditions to prevent contamination.
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 did 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

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 °C – 23.2°C

pH:

8.2

Dissolved oxygen:

no data

Salinity:

no data

Conductivity:

no data

Nominal and measured concentrations:

Control - 10- 32 – 102 – 328 and 1050 mg

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. 896 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 on the toxicological endpoint “inhibition of growth rate” indicate an ErC10-(0-72h) = 761 [713 - 799] mg/L , 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) 345 [265 - 437] mg/L, 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) = 896 mg/ L [845 - 945], value expressed in nominal test item concentration.

Results with reference substance (positive control):

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.

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 ErC10-(0-72h) = 761 [713 - 799] mg/L , 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) 345 [265 - 437] mg/L, 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) = 896 mg/ L [845 - 945], 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 algaPseudokirchneriella subcapitata, according to the OECD 201 guideline (March 2006, corrected July 2011).

The study was carried out in accordance with the study plan n°18/1136F/b dated from March 14, 2019.

There was no deviation during this study.

There was no amendment during this study.

The results obtained for thetest item on the toxicological endpoint “inhibition of growth rate” indicate an ErC₁₀-(0-72h) = 761 [713 - 799] mg/L ,value expressed in nominal test item concentration.

 

The results obtained for thetest 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)345 [265 - 437] mg/L,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)= 896 mg/ L [845 - 945],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.