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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2017-07-07 to 2017-08-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Principles of method if other than guideline:
Not relevant.
GLP compliance:
yes (incl. QA statement)
Remarks:
2017-01-10
Specific details on test material used for the study:
None.
Analytical monitoring:
yes
Vehicle:
yes
Remarks:
Use of solvent: acetone
Details on test solutions:
PREPARATION OF WAFs AND APPLICATION OF TEST SOLUTION
The study was carried out using WAFs (Water Accommodated Fractions). The WAFs were prepared under closed conditions and by slow-stirring.
The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of each mixing vessel was approximately 1 L. A magnetic stirring bar was placed in each mixing vessel completely filled with test water (with a minimum of headspace). The loading rates of the test item were weighed in glass vials and resuspended in acetone. Then the loading rates of the test item/vehicle were vortexed to obtain
homogeneous suspensions, and thereafter a specific volume (corresponding to a (limit) solvent concentration of 100 mg.L-1, as recommended in OECD guidance on testing and assessment No. 23) of each preparation was carefully added to the surface of test water contained in the mixing vessels that were closed immediately.
The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 23 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for at least 1 hour before use. The first 100 mL were removed via the drain port. Then the WAFs were directly added into test flasks containing a fixed amount of inoculum (5000 cells.mL-1 per vessel) that were immediately sealed after filling with a minimum of headspace. At the start of the test, the solution was observed to be clear and colourless.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata, CCAP 278/4
- Source (laboratory, culture collection): Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 -75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes under standardised conditions according to the test guidelines.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (=test water) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2°C.

ACCLIMATION
2 to 4 days before the start of the test, cells from the algal stock culture were inoculated in test water at a cell density of 10 000 cells.mL-1. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
None.
Post exposure observation period:
None.
Hardness:
No data.
Test temperature:
23°C ± 2°C
pH:
Not varying by more than 1.5 units at the end of the test in the control (8.20-9.98)
8.20-8.37 (start) - 8.34-10.01(end)
Dissolved oxygen:
No data.
Salinity:
No data.
Conductivity:
No data.
Nominal and measured concentrations:
Nominal concentration (mg test item.L-1): 0, 2.0, 4.4, 10.0, 22.3, 50.0
Details on test conditions:
TEST SYSTEM
Test vessel:
100 mL, all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace.
Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.

Replicates:
6 controls, 6 solvent controls and 3 replicates of each loading rate for counting. Moreover, a replicate of each treatment without algae was prepared for chemical analyses.

Initial cells density: 5 000 cells.mL-1 per vessel

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
The stock solutions 1 and 3 were sterilised by autoclaving (120 °C, 15 min), then stored in the dark at 4°C. The stock solutions 2 and 4 were sterilised by membrane filtration (mean pore diameter 0.2 μm). The growth medium was prepared by adding an appropriate volume of the stock solutions 1-4 to water.
To 500 mL of sterilised water were added:
- 10 mL of stock solution 1
- 1 mL of stock solution 2
- 1 mL of stock solution 3
- 1 mL of stock solution 4
Then, the mixture was made up to 1000 mL with sterilised water. The pH of this solution was approximately in the range of 8.1 ± 0.2.
Stock solution 1: macronutrients
NH4Cl 1.5 g.L-1
MgCl2·6H2O 1.2 g.L-1
CaCl2·2H2O 1.8 g.L-1
MgSO4·7H2O 1.5 g.L-1
KH2PO4 0.16 g.L-1
Stock solution 2: iron
FeCl3·6H2O 64 mg.L-1
Na2EDTA·2H2O 100 mg.L-1
Stock solution 3: trace elements
H3BO3 185 mg.L-1
MnCl2·4H2O 415 mg.L-1
ZnCl2 3 mg.L-1
CoCl2·6H2O 1.5 mg.L-1
CuCl2·2H2O 0.01 mg.L-1
Na2MoO4·2H2O 7 mg.L-1
Stock solution 4: bicarbonate
NaHCO3 50 g.L-1


OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination
- Light intensity and quality:4 440-8 880 lux


EFFECT PARAMETERS MEASURED:
Cell numbers were counted daily by microscope using a counting chamber.

TEST CONCENTRATIONS
Based on the results of a range-finding test, test solutions used in the definitive test were prepared to obtain the following loading rates (spaced by a factor of approximately 2.24): 2.0, 4.4, 10.0, 22.3 and 50.0 mg.L-1.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
2.786 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rates of test material
Basis for effect:
growth rate
Remarks on result:
other: CL95%: 2.222 – 3.326
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
9.819 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rates of test material
Basis for effect:
growth rate
Remarks on result:
other: CL95%: 8.919 – 10.808
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
0.972 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rates of test material
Basis for effect:
other: Yield
Remarks on result:
other: CL95%: 0.790 – 1.139
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
2.876 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rates of test material
Basis for effect:
other: Yield
Remarks on result:
other: CL95%: 2.673 – 3.082
Details on results:
Based on the results of the range-finding test, the effect of the test item on Pseudokirchneriella subcapitata was tested in a definitive study using five loading rates0, 2.0, 4.4, 10.0, 22.3, 50.0 mg/L.
Results with reference substance (positive control):
On February 14, 2017 (most recent test), the 72h-EC50 was 1.438 mg.L-1 for the parameter growth rate. Hence, the sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (expected 72h-EC50: 0.92 mg.L-1 to 1.46 mg.L-1).
Reported statistics and error estimates:
No data.

Analytical results

Concentration of dissolved organic material in the controls and the WAFs was checked by TOC analysis at the start and at the end of the test.

The analytical results of this test revealed that TOC analysis was not specific enough to separate organic carbon related to the solvent from the one of the test item. Besides, TOC analysis at the start and at the end of the study suggested that WAFs concentrations were overall stable, and indicated presence of organic compounds that was inversely proportional to the tested loading rates (i.e. TOC content was higher in the lowest loading rates) especially at the start of the test. Nevertheless, it cannot be ignored that test item presence in WAFs was undoubtedly proportional to the tested loading rates given the algal growth inhibition observed in the final test and the analytical test results (TOC content) without solvent during the range-finding test. It should be noted that a WAF is by definition a complex mixture for which the individual concentration of each constituent differs due to its properties (e.g. solubility, adsorption, volatilisation, bioaccumulation…). Due to these differences, interactions between certain constituents of the mixture may occur and affect the behaviour of a given constituent which consequently would not react in the same way that if it was alone in the mixture. Therefore, and since the test item was a UVCB substance, the results were based on the nominal test loading rates.

Biological results (see fig.1.)

Table: Algal cell densities during the final test (expressed as density of algal cells.mL-1x104)

  Replicate Nominal concentration
 (mg test item.L-1)*

Control

Solvent Control

2

4.4

10

22.3

50

t=24h

1

6.8

4.8

6

5.2

4.4

1.6

0.4

2

4.8

4.8

6

5.2

4.4

1.2

0.4

3

5.2

5.2

5.6

4.8

4.8

0.8

0.8

4

6.8

5.6

 

 

 

 

 

5

5.2

6

 

 

 

 

 

6

4.8

5.6

 

 

 

 

 

Mean

5.6

5.3

5.9

5.1

4.5

1.2

0.5

Std. Dev.

0.95

0.48

0.23

0.23

0.23

0.4

0.23

t=48h

1

24

20.8

23.2

13.2

9.6

1.6

0.4

2

21.2

26

22.8

14

6

0.8

0

3

23.2

23.2

24

11.6

5.6

1.2

0.8

4

26.4

24.4

 

 

 

 

 

5

21.2

28

 

 

 

 

 

6

23.6

26.4

 

 

 

 

 

Mean

23.3

24.8

23.3

12.9

7.1

1.2

0.4

Std. Dev.

1.95

2.57

0.61

1.22

2.2

0.4

0.35

t=72h

1

80.4

94.8

62

36

12

1.6

0

2

100

116.4

68.4

24

6.4

1.2

0

3

88

91.6

72.4

32

6.8

1.6

0

4

66.4

82.8

 

 

 

 

 

5

82.4

114.4

 

 

 

 

 

6

66.4

104.4

 

 

 

 

 

Mean

80.6

100.7

67.6

30.7

8.4

1.5

0

Std. Dev.

12.94

13.31

5.25

6.11

3.12

0.23

0

* WAF prepared at the given loading rate.

At test start 5000 algal cells.mL-1 were incubated; 6 replicates of the controls, 6 replicates of the solvent controls and 3 replicates of each test loading rate.

Std. Dev.: standard deviation.

Validity criteria of the study

Cell density in controls*      202-fold increase within 72 hours. The specific growth rate was determined at 1.77 day-1, so greater than 0.92 day-1.

Coefficient of variation       1. The mean coefficient of variation for section-by-section specific growth rates in the control cultures was of 29.7%.

2. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 2.5%.

*Since solvents were used to prepare the test solutions, the solvent controls rather than the controls without solvents were used in calculation of percent inhibition (as mentioned in OECD TG 201), and therefore for the validity criteria of the study.

Validity criteria fulfilled:
yes
Conclusions:
The toxic effect of the test item GALBANUM RESINOID to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a static limit test using Water Accommodated Fractions. Under the experimental conditions and based on nominal concentrations, 72h-EL50(growth) and 72h-EL50(yield) values were determined to be 9.819 and 2.876 mg test item.L-1 (loading rate), respectively.
Executive summary:

A study was performed to assess the test item GALBANUM RESINOID for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 amended by Commission Regulation (EU) 2016/266 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Following a preliminary range-finding test, algal cells were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 2.0, 4.4, 10.0, 22.3 and 50.0 mg.L-1 and to a control. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Concentration of dissolved organic material in the controls and the WAFs was checked by TOC analysis at the start and the end of the test.

The toxic effect of the test item GALBANUM RESINOID to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test using Water Accommodated Fractions. Under the experimental conditions and based on nominal concentrations, the 72-hour EL50 for the parameters growth rate and yield were determined to be 9.819 mg.L-1 and 2.876 mg.L-1, respectively. The 72-hour EL10 for growth rate was 2.786 mg L-1 and for yield was 0.972 mg.L-1.

Results synopsis:

72 -hour EL50 = 9.819 mg.L-1 (loading rate) based on parameter growth rate

72 -hour RL10 = 2.876 mg.L-1(loading rate) based on parameter growth rate

Description of key information

Based on experimental study,72-hour EL50 of GALBANUM RESINOID for the parameters growth rate and yield were determined to be 9.819 mg.L-1and 2.876 mg.L-1,respectively. The 72-hour EL10 for growth rate was 2.786 mg L-1and for yield was 0.972 mg.L-1.

Based on these results, the test item GALBANUM RESINOID would not be acutely toxic to aquatic organisms.

Key value for chemical safety assessment

EC50 for freshwater algae:
9.819 mg/L
EC10 or NOEC for freshwater algae:
2.786 mg/L

Additional information

A study was performed to assess the test item GALBANUM RESINOID for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 amended by Commission Regulation (EU) 2016/266 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Following a preliminary range-finding test, algal cells were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 2.0, 4.4, 10.0, 22.3 and 50.0 mg.L-1and to a control. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Concentration of dissolved organic material in the controls and the WAFs was checked by TOC analysis at the start and the end of the test.

The toxic effect of the test item GALBANUM RESINOID to the unicellular algal speciesPseudokirchneriella subcapitatawas investigated in a closed static test using Water Accommodated Fractions. Under the experimental conditions and based on nominal concentrations, the 72-hour EL50 for the parameters growth rate and yield were determined to be 9.819 mg.L-1and 2.876 mg.L-1,respectively. The 72-hour EL10 for growth rate was 2.786 mg L-1and for yield was 0.972 mg.L-1.

Based on these results, the test item GALBANUM RESINOID would not be acutely toxic to aquatic organisms.