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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:
19 May - 16 Jul 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples were taken from the control (replicates R1-R6 pooled) and each test group (replicates R1-R3 pooled) at 0 and 72 h.
- Sample storage conditions before analysis: Stored at approx. -20 °C for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item solutions were prepared by stirring an excess (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Gelman Acrocap filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a saturated solution of the test item with a nominal concentration of 50 mg/L. This saturated solution was then further diluted as necessary, to provide the remaining test groups.
- Differential loading: no
- Controls: yes
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Method of cultivation: Maintained in the laboratory under constant aeration and constant illumination at 21 +/- 1 °C. Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10³ cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 - 10^5 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ± 1 °C
pH:
7.0 - 7.2 (beginning of the study)
7.3 - 7.8 (end of the study)
Nominal and measured concentrations:
Nominal concentrations: 0.0050, 0.016, 0.050, 0.16 and 0.50 mg/L
Measured concentrations: 0.0032, 0.0010, 0.037, 0.12 and 0.38 mg/L (0 h)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks (plugged with polyurethane foam bungs)
- Material, size, fill volume: glass, 250 mL, 100 mL
- Aeration: constantly shaken (150 rpm)
- Initial cells density: nominal cell density of 4x10³ cells/mL
- Control end cells density: 1.55E+05 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis purified deionised water and the pH adjusted to 7.5 +/- 0.1 N NaOH or HCl.

OTHER TEST CONDITIONS
- Photoperiod: constant illumination
- Light intensity and quality: warm white lighting (380 - 730 nm)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell concentrations were determined for each control and treatment group, using a Coulter® Multisizer Particle Counter (0, 24, 48 and 72 h)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: control, 0.050, 0.50, 5.0 and 50 mg/L (2 replicates)
- Results used to determine the conditions for the definitive study: 98 - 105% inhibtion of growth for the three highest test concentrations (0.5, 5.0, 50 mg/L) in the first range-finding test and 0 - 19% in the second range-finding test (0.0005, 0.005, 0.05 mg/L).
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.004 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.009 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.007 - 0.010 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.01 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.039 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.03 - 0.051 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.004 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.005 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 0.0050 - 0.0060 mg/L
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Much cell debris was observed to be present in the test cultures at 0.38 mg/L.
- Any stimulation of growth found in any treatment: no
- Effect concentrations exceeding solubility of substance in test medium: No, the solubility of the test substance was determined to be approx. 50 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.74 mg/L (growth rate) and 0.37 mg/L (yield)
- Other: NOEC: 0.25 (growth rate)
Reported statistics and error estimates:
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) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS Computer software package (SAS 1999 -2001). For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). EC values were then determined from the equation for the fitted line. Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

The study resulted in an EC50 of 0.039 mg/L based on the measured (initial) concentration. A NOEC was determined to be 0.01 mg/L (measured initial). The results are based on the initial measured concentrations because the geometric mean (0 h and 72 h) may overestimate the effects because the concentration in the test vessels was only measured at the beginning and at the end of the study.

 

Table 1: Geometric mean measured test concentrations

0-Hour Measured Test Concentration [mg/L]

Geometric Mean Measured Test Concentration [mg/L]

Expresses as a % of the 0-Hour Measured Test Concentration

0.0032

0.0022

69

0.010

0.0044

44

0.037

0.0077

21

0.12

0.021

18

0.38

0.22

58

Table 2: Inhibition of growth rate

0-Hour Measured Test concentration [mg/L]

Growth Rate [cells/mL/hour]

0 – 72 h

% inhibition

Control

R1

0.050

-

R2

0.053

-

R3

0.048

-

R4

0.053

-

R5

0.049

-

R6

0.050

-

Mean

0.051

-

SD

0.002

-

0.0032

R1

0.049

4

R2

0.046

10

R3

0.058

[14]

Mean

0.051

0

SD

0.0060

 

0.010

R1

0.041

20

R2

0.043

16

R3

0.043

16

Mean

0.042

17

SD

0.001

 

0.037

R1

0.019

63

R2

0.025

51

R3

0.044

14

Mean

0.029

43

SD

0.013

 

0.12

R1

0.009

82

R2

0.008

84

R3

-0.001

102

Mean

0.005

89

SD

0.006

 

0.38

R1

-0.009

118

R2

-0.005

110

R3

-0.005

110

Mean

-0.006

113

SD

0.002

 

Values in square brackets indicates an increase of growth

Description of key information

NOEC (72 h): 0.0044 mg/L (measured, geometric mean) for Desmodesmus subspicatus (OECD 201)
EC50 (72 h): 0.0087 mg/L (measured, geometric mean) for Desmodesmus subspicatus (OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
0.005 mg/L
EC10 or NOEC for freshwater algae:
0.004 mg/L

Additional information

The toxicity of Atmer 163 (CAS No. 97925-95-6) towards aquatic algae was investigated following the OECD Guideline 201 under GLP conditions (Vryenhoef, 2010). Desmodesmus subspicatus was used for testing and exposed to the test substance for 72 h in a static test. Nominal concentrations of 0.005, 0.016, 0.050, 0.16 and 0.50 mg/L were used in the test. Since the substance was not stable during testing the effects concentrations were based on the measured geometric mean concentration. After the exposure period of 72 h an EC50 of 0.0051 and a NOEC of 0.0044 mg/L based on the growth rate were determined.