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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:
06 September 2016 to ****
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
CAS RN: 15337-18-5
Purity: 96.6%
Physical state/Appearance: Yellow viscous liquid
Analytical monitoring:
yes
Details on sampling:
Samples were taken in duplicate from the control and each treatment group at 0 and 72 hours and from samples run alongside the test at 24 and 48 hours for quantitative analysis. All samples were stored frozen prior to analysis.

Preparation of Test Samples
The test samples were thawed with the aid of sonication before being filtered through 0.45 µm cellulose acetate filters.
Vehicle:
no
Details on test solutions:
Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation. A preliminary media preparation trial indicated that a saturated solution method of preparation was the most appropriate for this test item. Based on the results of a preliminary range-finding test and an initial experiment, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 3.13, 6.25, 12.5, 25, 50 and 100% v/v saturated solution (3 replicate flasks per concentration) for 72 hours. A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorious Sartopore filter (first approximate 2 liters discarded to pre condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to achieve definitive test concentrations of 3.13, 6.25, 12.5, 25 and 50% v/v saturated solution.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 2 ºC.
Test type:
static
Water media type:
other: culture medium
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ±1 ºC
pH:
The pH value of the control cultures was observed to increase from pH 7.2 at 0 hours to pH 8.5 at 72 hours.
Nominal and measured concentrations:
Nominal: 3.13, 6.25, 12.5, 25, 50 and 100% v/v saturated solution.
Measured: Chemical analysis of the 10 and 100% v/v saturated solution test preparations at 0 and 72 hours showed measured concentrations of zinc to range from less than the limit of quantitation (LOQ) of the analytical method employed (0.010 mg/L), to 0.017 mg/L, equivalent to concentrations of less than the LOQ to 0.159 mg/L as test item.
Details on test conditions:
As in the range-finding test and initial experiment, 250 mL glass conical flasks were used in the definitive test. Six flasks each containing 100 mL of culture medium were used for the control and three flasks each containing 100 mL of test solution were used for each treatment group.

The control group was maintained concurrently under identical conditions, but was not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 7.57 x 10^5 cells per mL. Inoculation of 900 mL of test medium with 5.9 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated at 24 ±1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Samples were taken at 23, 45 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Growth rate, yield and biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12.5 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.25 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield and Biomass
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
25 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
12.5 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield and Biomass
Details on results:
Verification of Test Concentrations
Analysis of the test preparations at 0, 24, 48 and 72 hours to confirm test item concentrations was based on the quantitation of elemental zinc and back-calculation of total substance. Measured concentrations in the definitive test were less than the LOQ of the analytical method employed were obtained, which was determined to be 0.010 mg/L (equivalent to 0.093 mg/L as test item).

Given that the toxicity in this study cannot be attributed to a single elemental component, but to the dissolved fraction of the total test item and free zinc, which was below the quantifiable limit of the analytical method, the results were based on nominal concentrations as % v/v saturated solution.

Growth Data
The growth rate (r), yield (y) and biomass (b) of Pseudokirchneriella subcapitata (CCAP 278/4) were inhibited by the presence of the test item over the 72 hour exposure period.

Inhibition of Growth Rate
ErC10 (0 to 72 hour): >100% v/v Saturated Solution
ErC20 (0 to 72 hour): >100% v/v Saturated Solution
ErC50 (0 to 72 hour): >100% v/v Saturated Solution

The NOEC based on growth rate was determined to be 12.5% v/v saturated solution and the Lowest Observed Effect Concentration (LOEC) based on growth rate was 25% v/v saturated solution.

Inhibition of Yield
EyC10 (0 to 72 hour): 12% v/v Saturated Solution
EyC20 (0 to 72 hour): 32% v/v Saturated Solution
EyC50 (0 to 72 hour): >100% v/v Saturated Solution

The NOEC based on yield was determined to be 6.25% v/v saturated solution and the LOEC based on yield was 12.5% v/v saturated solution.

Inhibition of Biomass
EbC10 (0 to 72 hour): 11% v/v Saturated Solution
EbC20 (0 to 72 hour): 29% v/v Saturated Solution
EbC50 (0 to 72 hour): >100% v/v Saturated Solution

The NOEC based on biomass was therefore determined to be 6.25% v/v saturated solution and the LOEC based on biomass was 12.5% v/v saturated solution.
Results with reference substance (positive control):
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item, potassium dichromate, gave the following results:

ErC50 (0 – 72 h): 1.6 mg/L; 95% confidence limits 1.4 – 1.8 mg/L
EyC50 (0 – 72 h): 0.77 mg/L; 95% confidence limits 0.68 – 0.87 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
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). There were no statistically significant differences in growth rate between the control and the 3.13, 6.25 and 12.5% v/v saturated solutions (P≥0.05). Inhibition of growth rate observed at 50 and 100% v/v saturated solutions was significant compared to the controls (P<0.05), however.

There were no statistically significant differences in yield or biomass between the control and the 3.13 and 6.25% v/v saturated solution test concentrations (P≥0.05). Inhibition of yield and biomass observed at 12.5, 25, 50 and 100% v/v saturated solutions was significant compared to the controls (P<0.05), however.

Inhibition of Growth Rate, Yield and Biomass in the Definitive Test

Nominal Concentration

Growth Rate (cells/mL/hour)

Yield (cells/mL)

Biomass Integral

(% v/v Saturated Solution)

0 to 72 Hour

% Inhibition

0 to 72 Hour

% Inhibition*

0 - 72 h

% Inhibition

Control

R1

0.079

 

1.44E+06

 

2.12E+07

 

R2

0.079

 

1.44E+06

 

2.11E+07

 

R3

0.079

 

1.44E+06

 

2.10E+07

 

R4

0.078

-

1.42E+06

-

2.03E+07

-

R5

0.079

 

1.51E+06

 

2.18E+07

 

R6

0.079

 

1.52E+06

 

2.18E+07

 

Mean

0.079

 

1.46E+06

 

2.12E+07

 

SD

0

 

4.38E+04

 

5.41E+05

 

3.13

R1

0.08

[1]

1.53E+06

 

2.30E+07

[8]

R2

0.079

0

1.50E+06

 

2.15E+07

[2]

R3

0.079

0

1.50E+06

 

2.14E+07

[1]

Mean

0.079

[0]

1.51E+06

[3]

2.20E+07

[4]

SD

0.001

 

1.84E+04

 

8.67E+05

 

6.25

R1

0.079

0

1.44E+06

 

2.08E+07

2

R2

0.079

0

1.49E+06

 

2.13E+07

0

R3

0.079

0

1.45E+06

 

2.06E+07

3

Mean

0.079

0

1.46E+06

0

2.09E+07

SD

0

 

2.37E+04

 

3.77E+05

 

12.5

R1

0.078

1

1.33E+06

 

1.89E+07

11

R2

0.078

1

1.35E+06

 

1.96E+07

8

R3

0.077

3

1.24E+06

 

1.78E+07

16

Mean

0.078

2

1.31E+06

11

1.87E+07

12

SD

0.001

 

5.73E+04

 

9.30E+05

 

25

R1

0.076

4

1.18E+06

 

1.69E+07

20

R2

0.076

4

1.17E+06

 

1.71E+07

19

R3

0.076

4

1.15E+06

 

1.64E+07

23

Mean

0.076

4

1.17E+06

20

1.68E+07

21

SD

0

 

1.35E+04

 

4.06E+05

 

50

R1

0.075

5

1.08E+06

 

1.52E+07

28

R2

0.073

8

9.38E+05

 

1.31E+07

38

R3

0.072

9

8.58E+05

 

1.22E+07

42

Mean

0.073

7

9.59E+05

34

1.35E+07

36

SD

0.002

 

1.13E+05

 

1.54E+06

 

100

R1

0.074

6

1.06E+06

 

1.48E+07

30

R2

0.072

9

8.96E+05

 

1.24E+07

41

R3

0.073

8

9.45E+05

 

1.32E+07

38

Mean

0.073

8

9.68E+05

34

1.35E+07

36

SD

0.001

 

8.57E+04

 

1.20E+06

 

* = In accordance with the OECD test guideline 201, only the mean value for yield for each test concentration is calculated

R = Replicate

SD = Standard Deviation

[ ] = increase in growth rate compared to the controls

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata was investigated and the 72 hour EC50 value based on growth rate was determined to be greater than 100% v/v saturated solution, the NOEC was given as 12.5% v/v saturated solution and the LOEC determined to be 25% v/v saturated solution.
Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation (EC) No 761/2009.

 

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation. A preliminary media preparation trial indicated that a saturated solution method of preparation was the most appropriate for this test item. Based on the results of a preliminary range-finding test and an initial experiment, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 3.13, 6.25, 12.5, 25, 50 and 100% v/v saturated solution (3 replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 ºC. Samples of the solutions were removed daily and cell concentrations were determined for each control and treatment group.

 

Test solutions were collected at 0, 24, 48 and 72 hours and analyzed by inductively coupled plasma with mass spectrometric detection (ICP-MS) to confirm test item concentrations based on the quantitation of elemental zinc. Measured concentrations in the definitive test were less than the Limit of Quantification (LOQ) of the analytical method employed, which was determined to be 0.010 mg/L zinc (equivalent to 0.093 mg/L as test item). Given that the toxicity in this study cannot be attributed to a single elemental component, but to the dissolved fraction of the total test item and free zinc, which was below the quantifiable limit of the analytical method, the results were based on nominal concentrations as % v/v saturated solution.

 

In conclusion, the effect of the test item, on the growth of Pseudokirchneriella subcapitata was investigated over a 72-hour period and based on the nominal test concentrations gave the following results based on the nominal test concentrations:

 

Response Variable

EC50
(% v/v Saturated Solution)

NOEC
(% v/v Saturated Solution)

LOEC
(% v/v Saturated Solution)

Growth Rate

>100

12.5

25

Yield

>100

6.25

12.5

Biomass

>100

6.25

12.5

 

 

Description of key information

The effect of the test item on the growth of Pseudokirchneriella subcapitata was investigated and the 72 hour EC50 value based on growth rate was determined to be greater than 100% v/v saturated solution, the NOEC was given as 12.5% v/v saturated solution and the LOEC determined to be 25% v/v saturated solution.

Key value for chemical safety assessment

Additional information