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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:
17 May 2016 to 22 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours. Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for immediate quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
A nominal amount of test item (500 mg) was dispersed in 5 liters of culture medium with the aid of ultrasonication for approximately 10 minutes. After sonicating any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.
A series of dilutions was made from this saturated solution to give further stock solutions of 32, 10, 3.2 and 1.0% v/v saturated solution. An aliquot (1800 mL) of each of the stock solutions was separately inoculated with 37 mL of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution. The stocks and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 2 °C.



Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ± 1 °C
Nominal and measured concentrations:
nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL glass conical flasks each completely filled with test preparation and sealed with ground glass stoppers to reduce evaporation
- Aeration: No
- Initial cells density: 5.00 x E+03 cells/mL
- Control end cells density: 5.65 x E+05 cells/mL
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2


GROWTH MEDIUM
- Standard medium used: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

TEST MEDIUM / WATER PARAMETERS
The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.
For the purposes of the range-finding and definitive tests, additional sodium bicarbonate (250 mg/L) was added to the prepared culture medium prior to use and to prevent inhibition of growth due to the restriction in gaseous exchange associated with testing in an enclosed system (Herman et al., 1990).

- NaNO3: 25.5 mg/L
- MgCl2 x 6H2O: 12.16 mg/L
- CaCl2 x 2H2O: 4.41 mg/L
- MgSO4.7H2O 14.6 mg/L
- K2HPO4: 1.044 mg/L
- NaHCO3: 15.0 mg/L
- H3BO3: 0.186 mg/L
- MnCl2 x 4H2O 0.415 mg/L
- ZnCl2: 0.00327 mg/L
- FeCl3 x 6H2O: 0.160 mg/L
- CoCl2 x 6H2O: 0.00143 mg/L
- Na2MoO4 x 2H2O: 0.00726 mg/L
- CuCl2 x 2H2O: 0.000012 mg/L
- Na2EDTA x 2H2O: 0.30 mg/L


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 7000 lux provided by warm white lighting (380 – 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.63 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): After 72 hours there were no abnormalities detected in the control or test cultures at 0.30, 0.80 and 4.2 mg/L, however no intact cells were observed to be present in the test cultures at 12 and 49 mg/L.
- Colour differences: At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-hour test period all control, 0.30 and 0.80 mg/L test cultures were observed to be green dispersions. The 4.2 mg/L test cultures were extremely pale green dispersions whilst the 12 and 49 mg/L test cultures were clear colorless solutions.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 1.5 mg/L; 95% confidence limits 1.3 – 1.7 mg/L
Reported statistics and error estimates:
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955).

Table 1. Cell Densities and pH Values in the Definitive Test

Geometric Mean Measured Test Concentration [mg/l] pH Cell density* (cells per mL) pH
0 h 23 h 48 h 72 h 72 h
Control R1 7.4 2.17E+04 1.72E+05 5.60E+05 9.8
R2 1.75E+04 1.65E+05 5.64E+05
R3 1.95E+04 1.58E+05 5.62E+05
R4 1.26E+04 1.71E+05 6.47E+05
R5 1.90E+04 1.55E+05 5.06E+05
R6 1.17E+04 1.45E+05 5.49E+05
Mean 1.70E+04 1.61E+05 5.65E+05
0.3 R1 7.5 1.36E+04 1.70E+05 5.75E+05 9.7
R2 9.56E+03 1.70E+05 5.70E+05
R3 1.33E+04 1.65E+05 5.65E+05
Mean 1.22E+04 1.68E+05 5.70E+05
0.8 R1 7.6 7.16E+03 7.27E+04 3.00E+05 9.4
R2 8.83E+03 8.05E+04 3.68E+05
R3 9.56E+03 6.80E+04 3.18E+05
Mean 8.52E+03 7.37E+04 3.29E+05
4.2 R1 7.6 6.01E+03 1.31E+04 1.83E+04 8.5
R2 6.98E+03 1.41E+04 2.03E+04
R3 7.30E+03 1.23E+04 2.93E+04
Mean 6.77E+03 1.31E+04 2.26E+04
12 R1 7.6 7.25E+03 8.18E+03 1.05E+04 8.1
R2 6.83E+03 8.80E+03 1.25E+04
R3 9.62E+03 8.65E+03 9.62E+03
Mean 7.90E+03 8.55E+03 1.09E+04
49 R1 7.7 7.36E+03 7.16E+03 6.75E+03 8.1
R2 7.80E+03 7.42E+03 5.69E+03
R3 8.51E+03 7.10E+03 6.89E+03
Mean 7.89E+03 7.23E+03 6.44E+03
* Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks. 
R1 - R6 = Replicates l to 6

Table 2. Daily Specific Growth Rates for the Control Cultures in the Definitive Test

Daily Specific Growth Rate (cells/mL/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

Control

R1

0.064

0.083

0.049

R2

0.055

0.09

0.051

R3

0.059

0.084

0.053

R4

0.04

0.104

0.055

R5

0.058

0.084

0.049

R6

0.037

0.101

0.056

Mean

0.052

0.091

0.052

Table 3. Inhibition of Growth Rate

Geometric Mean Measured Test Concentration [mg/l]

Cell density*

(cells per mL)

0-72h

% Inhibition

Control

R1

0.066

R2

0.066

R3

0.066

R4

0.068

R5

0.064

R6

0.065

Mean

0.066

SD

0.001

0.3

R1

0.066

0

R2

0.066

0

R3

0.066

0

Mean

0.066

0

SD

0

0.8

R1

0.057

14

R2

0.06

9

R3

0.058

12

Mean

0.058

12

SD

0.002

4.2

R1

0.018

73

R2

0.019

71

R3

0.025

62

Mean

0.021

69

SD

0.004

12

R1

0.01

85

R2

0.013

80

R3

0.009

86

Mean

0.011

84

SD

0.002

49

R1

0.004

94

R2

0.002

97

R3

0.004

94

Mean

0.003

95

SD

0.001

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R1 – R6 = Replicates 1 to 6

SD = Standard Deviation

[Increase in growth as compared to controls]

The results of the test are considered valid since the following performance criteria are met:

1.) The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.

--> The study data show that the cell concentration of the control cultures increased by a factor of 113 after 72 hours.

2.) The mean of the coefficients of variation of the section by section daily growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3) must not exceed 35%.

--> The mean coefficient of variation for section by section specific growth rate for the control cultures was 35% and hence satisfied the validation criterion given in the OECD Guideline.

3.) The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.

--> The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 2% and hence satisfied the validation criterion given in the OECD Guideline.

Validity criteria fulfilled:
yes
Conclusions:
In a 72-hour acute toxicity study reported by Vryenhoef (2016), cultures of Pseudokirchneriella subcapitata were exposed to (3E)-dec-3-en-2-one at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution under static conditions in accordance with OECD guideline 201. The effect of (3E)-dec-3-en-2-one on the growth of P. subcapitata based on the geometric mean measured test concentrations gave the following results:
- ErC10: 0.63 mg/L
- ErC50: 2.8 mg/L (95%-C.I.: 2.1 - 3.8 mg/L);
- LOErC: 0.8 mg/L;
- NOErC: 0.3 mg/L.
The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any abnormalities.
This toxicity study is classified as acceptable and satisfies the guideline requirements for algae growth inhibition study according to OECD guideline 201.
Executive summary:

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata (CCAP 278/4) were exposed to (3E)-dec-3-en-2-one at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution under static conditions in accordance with the OECD 201 guideline. The NOEC and EC50 values based on growth rate were 0.3 g/L and 2.8 mg/L, respectively.

There were no compound related phytotoxic effects.

This toxicity study is classified as acceptable and satisfies the guideline requirements for alga growth inhibition study with P. subcapitata.

Results synopsis

Test organism: Pseudokirchneriella subcapitata

Test type: Static

All effect values are reported as geometric means of measured concentrations:

- 72-h ErC10: 0.63 mg/L;

- 72 -h ErC20: 1.1 mg/L;

- 72 -h ErC50: 2.8 mg/L (95% confidence limits 2.1 – 3.8 mg/L);

- NOErC: 0.3 mg/L;

- LOErC: 0.8 mg/L.

Endpoint(s) effected: Growth inhibition, abnormalities of the cells

Description of key information

In a 72-hour acute toxicity study reported by Vryenhoef (2016), cultures of Pseudokirchneriella subcapitata were exposed to (3E)-dec-3-en-2-one at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution under static conditions in accordance with OECD guideline 201. The effect of (EC)-dec-3-en-2-one

on the growth of Pseudokirchneriella subcapitata based on the geometric mean measured test concentrations gave the following results:

- ErC10: 0.63 mg/L

- ErC50: 2.8 mg/L (95%-C.I.: 2.1 - 3.8 mg/L);

- LOErC: 0.8 mg/L;

- NOErC: 0.3 mg/L.

The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any abnormalities.

This toxicity study is classified as acceptable and satisfies the guideline requirements for algae growth inhibition study according to OECD guideline 201.

Key value for chemical safety assessment

EC50 for freshwater algae:
2.8 mg/L
EC10 or NOEC for freshwater algae:
0.3 mg/L

Additional information