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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:
7/10/2013 - 9/10/2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to international guideline (OECD guideline 201) under GLP. No deviations from guideline reported.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Principles of method if other than guideline:
Not relevant
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not relevant
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1.0, 3.2, 10, 32 and 100 mg/L
- Sampling method: Samples were taken from the uninoculated control and each loading rate WAF test group at 0 and 72 hours for this analysis.
- Sample storage conditions before analysis: stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test item (22.5, 72, 22.3, 71.2 and 234 mg) were each separately added to the surface of 22.5, 22.5, 2.23, 2.23 and 2.34 liters of culture medium to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the media surface. The stirring was stopped after 47 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs. The test item was weighed out under non-actinic light, and the preparations were shielded from light during the mixing period due to the light-sensitive nature of the test item. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
- Controls: Yes, blanks
- Evidence of undissolved material (e.g. precipitate, surface film, etc): At both the start and the end of stirring, and following a 1-hour standing period, the 1.0, 3.2, 10, 32 and 100 mg/L WAFs were observed to be clear colourless water columns with an oily layer of test item on the surface. Microscopic observations of the WAFs after siphoning showed no microscopic particles to be present.

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-h test period all control, 1.0, 3.2 and 10 mg/L loading rate WAF test preparations were observed to be green dispersions. The 32 mg/L loading rate test preparations were observed to be extremely pale green dispersions and the 100 mg/L WAF test preparations were observed to be clear colourless solutions.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchnerella 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. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium.
- Method of cultivation: The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1ºC.

ACCLIMATION
- Culturing media and conditions (same as test or not): Yes, but range-finding and definitive test media contain 500 mg/L sodium bicarbonate to counteract the increase of pH. 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 10^3 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 ± 1ºC 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
Post exposure observation period:
Not relevant
Hardness:
No data
Test temperature:
24 +/- 1ºC
pH:
7.7 - 9.7 in control vessels, 6.7 - 9.7 in treated vessels (maximum increase of 2.5 during test)
Dissolved oxygen:
Not relevant
Salinity:
Not relevant
Nominal and measured concentrations:
Nominal: 1.0, 3.2, 10, 32 and 100 mg/L
Measured (t=0h): 0.17, 1.02, 1.50, 4.91 and 36.84 mg/L
Measured (t=72h): 0.50, 2.11, 0.95, 6.81 and 36.87 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 300 mL volume filled with 300 mL test medium
- Initial cells density: 6.05*10^3 cell/mL
- Control end cells density: 6.68x10^5
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:
NaNO3 25.5 mg/L
MgCl2.6H2O 12.164 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.7 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.1855 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.159 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionized water
- Culture medium different from test medium: No, but 500 mg/L sodium bicarbonate was added to test solutions to counteract the increase of pH during the test.
- Intervals of water quality measurement: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Photoperiod: continuous
- Light intensity and quality: warm white lighting (380 – 730 nm) at 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations:10 and 100 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
34 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95%CI: 29 - 39 mg/L
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
28 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95%CI: 27 - 29 mg/L
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0, 3.2 and 10 mg/L loading rate WAF, however no intact cells were observed to be present in the test cultures at 32 and 100 mg/L loading rate WAF.
- Any stimulation of growth found in any treatment: yes, in the 1.0 and 3.2 mg/L test vessels.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At both the start and the end of stirring, and following a 1-hour standing period, the 1.0, 3.2, 10, 32 and 100 mg/L WAFs were observed to be clear colourless water columns with an oily layer of test item on the surface. Microscopic observations of the WAFs after siphoning showed no microscopic particles to be present. At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-h test period all control, 1.0, 3.2 and 10 mg/L loading rate WAF test preparations were observed to be green dispersions. The 32 mg/L loading rate test preparations were observed to be extremely pale green dispersions and the 100 mg/L WAF test preparations were observed to be clear colourless solutions.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50: 1.1 mg/L
- EbC50: 0.51 mg/L
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 loading rates 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).

Cell densities and pH-values:

Nominal Loading Rate

(mg/L)

pH

Cell Densities*(cells per mL)

pH

0 h

0 h

24 h

46 h

72 h

72 h

Control

 

 

 

 

 

R1

7.7

5.13E+03

1.61E+04

1.24E+05

6.95E+05

9.7

R2

6.37E+03

1.67E+04

1.10E+05

7.53E+05

R3

6.16E+03

1.40E+04

9.32E+04

6.97E+05

R4

6.57E+03

1.59E+04

8.79E+04

5.97E+05

R5

5.37E+03

1.59E+04

1.05E+05

6.86E+05

R6

6.69E+03

1.43E+04

9.04E+04

5.81E+05

Mean

6.05E+03

1.55E+04

1.02E+05

6.68E+05

1.0

 

 

 

R1

7.2

6.04E+03

1.60E+04

1.32E+05

8.96E+05

9.7

R2

4.08E+03

1.17E+04

9.80E+04

6.70E+05

R3

5.43E+03

1.26E+04

1.04E+05

6.02E+05

Mean

5.18E+03

1.34E+04

1.11E+05

7.23E+05

3.2

 

 

 

R1

7.3

4.49E+03

1.53E+04

1.12E+05

7.95E+05

9.7

R2

3.87E+03

1.52E+04

1.10E+05

8.38E+05

R3

4.46E+03

1.35E+04

8.48E+04

6.74E+05

Mean

4.27E+03

1.47E+04

1.03E+05

7.69E+05

10

 

 

 

R1

7.1

2.43E+04

1.84E+04

1.27E+05

7.39E+05

9.6

R2

5.84E+03

1.32E+04

8.95E+04

8.14E+05

R3

5.84E+03

1.23E+04

9.17E+04

6.81E+05

Mean

1.20E+04

1.46E+04

1.03E+05

7.44E+05

32

 

 

 

R1

6.9

6.34E+03

8.54E+03

2.03E+04

5.92E+04

7.9

R2

5.10E+03

8.57E+03

1.70E+04

5.05E+04

R3

6.63E+03

8.68E+03

2.23E+04

1.21E+05

Mean

6.02E+03

8.59E+03

1.98E+04

7.68E+04

100

 

 

 

R1

6.7

7.66E+03

5.81E+03

6.01E+03

7.10E+03

7.5

R2

5.84E+03

5.60E+03

7.48E+03

7.22E+03

R3

5.66E+03

6.60E+03

7.10E+03

6.13E+03

Mean

6.38E+03

6.00E+03

6.86E+03

6.82E+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.

** No values available due to breakage of replicate prior to sampling at 48 hours.

R1- R6= Replicates 1 to 6

Daily specific growth rates in controls:

Daily Specific Growth Rate (cells/mL/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

Control

R1

0.051

0.082

0.072

R2

0.052

0.075

0.080

R3

0.045

0.076

0.084

R4

0.050

0.068

0.080

R5

0.050

0.075

0.078

R6

0.046

0.074

0.078

Mean

0.049

0.075

0.079

Inhibition of growth rate and yield:

Nominal Loading Rate
(mg/L)

Growth Rate

(cells/mL/hour)

Yield

(cells/mL)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

 

 

 

 

 

 

 

R1

0.069

 

6.90E+05

 

R2

0.070

 

7.46E+05

 

R3

0.069

 

6.91E+05

 

R4

0.066

-

5.91E+05

-

R5

0.068

 

6.81E+05

 

R6

0.066

 

5.74E+05

 

Mean

0.068

 

6.62E+05

 

SD

0.002

 

6.61E+04

 

1.0

 

 

 

 

R1

0.072

[6]

8.90E+05

 

R2

0.068

0

6.66E+05

 

R3

0.067

1

5.97E+05

 

Mean

0.069

[2]

7.18E+05

[8]

SD

0.003

 

1.53E+05

 

3.2

 

 

 

 

R1

0.070

[3]

7.91E+05

 

R2

0.071

[4]

8.34E+05

 

R3

0.068

0

6.69E+05

 

Mean

0.070

[2]

7.65E+05

[15]

SD

0.002

 

8.54E+04

 

10

 

 

 

 

R1

0.069

[1]

7.14E+05

 

R2

0.071

[4]

8.08E+05

 

R3

0.068

0

6.75E+05

 

Mean

0.069

[2]

7.32E+05

[1]

SD

0.002

 

6.84E+04

 

32

 

 

 

 

R1

0.034

50

5.28E+04

 

R2

0.032

53

4.54E+04

 

R3

0.044

35

1.14E+05

 

Mean

0.037

46

7.08E+04

89

SD

0.006

3.77E+04

 

100

 

 

 

R1

0.005

93

-5.57E+02

 

R2

0.005

93

1.38E+03

 

R3

0.003

96

4.69E+02

 

Mean

0.004

94

4.30E+02

100

SD

0.001

 

9.69E+02

 

*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]

Growth curves are shown below.

Validity criteria fulfilled:
yes
Remarks:
> 16-fold increase in controls, mean of coefficients of variation < 35%, specific coefficient of variation < 7%
Conclusions:
The ecotoxicity (72h-NOErL and 72h-ErL50) of Cymbopogon winterianus towards Pseudokirchnerella subcapitata is 34 and 10 mg/L respectively.
Executive summary:

The ecotoxicity of Cymbopogon winterianus towards Pseudokirchnerella subcapitata was investigated according to OECD guideline 201 under GLP. Algal cells were exposed to WAFs with nominal loading rates of 1.0. 3.2, 10, 32 and 100 mg/L and were observed for 72 hours. The 72h-NOErL and 72h-ErL50 were found to be 34 and 10 mg/L respectively based on nominal concentrations.

Description of key information

The 72h-NOErL and 72h-ErL50 are 10 and 34 mg/L respectvely in an OECD guideline 201 test with Pseudokirchnerella subcapitata under GLP (reliability 1).

Key value for chemical safety assessment

EC50 for freshwater algae:
34 mg/L
EC10 or NOEC for freshwater algae:
10 mg/L

Additional information

The ecotoxicity of Cymbopogon winterianus towards Pseudokirchnerella subcapitata was investigated according to OECD guideline 201 under GLP. Algal cells were exposed to WAFs with nominal loading rates of 1.0. 3.2, 10, 32 and 100 mg/L and were observed for 72 hours. The 72h-NOErL and 72h-ErL50 were found to be 34 and 10 mg/L respectively based on nominal concentrations.