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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:
23 July 2012 to 26 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
The temperature of the test medium was below the range specified by the guidelines by 1ºC
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
The temperature of the test medium was below the range specified by the guidelines by 1ºC
Qualifier:
according to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
not specified
Principles of method if other than guideline:
Deviation from guidelines: The temperature of the test medium was below the range specified by the guidelines by 1ºC. The lower temperature was only for a short period at the start of the study, as the incubator regulated the temperature within the optimum range throughout the remainder of the study. As such, this deviation is not considered to have had an adverse effect on algal growth.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Single samples from all test concentration and the control were collected.
- Sampling method: 2 mL samples were collected at 0, 24 and 72 hrs post application. At the end of exposure the replicates containing algae were pooled at each concentration before sampling. Reserve samples were also collected and stored for future analysis if required.
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with the highest concentration of 100 mg/L applying 21 minutes of magnetic stirring to ensure complete dissolving of the test material in the test medium. The lower test concentrations (combined range-find test) were prepared by subsequent dilutions of the highest concentration in test medium.
- Controls: An untreated blank medium control as run concurrently.
- Evidence of undissolved material: The final test solutions were all clear and colourless.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: NIVA CHL 1
- Source: In house laboratory culture.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (light intensity, 60 to 120 µE/m2/s) at a room temperature of 21-24°C.

ACCLIMATION
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/ml. The cell density was measured immediately before use.
- Culturing media and conditions: The pre-culture was maintained under the same conditions as used in the test. Pre-culture medium and stock culture medium compositions can be seen in Table 1.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
none
Test temperature:
The temperature measured 20 ºC at the beginning of the test and was then maintained between 23.0 and 23.3 ºC throughout the remainder of the test.
pH:
Control: 8.3 at 0 hours and 8.0 at 72 hours.
100 mg/L: 8.2 at 0 hours and 8.0 at 72 hours.
Nominal and measured concentrations:
Nominal concentration of 100 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass.
- Volume of test solution: 50 mL of the appropriate test solution was added to each vessel.
- Aeration:
- Initial cells density: 1 x 10^4 cells/mL, equivalent to 1 mL of algal suspension.
- Incubation: Test vessels were placed in a shaking incubator in order to keep cells in suspension.
- No. of vessels per concentration: 6 replicates, one extra replicate was made for sampling after 24 hours.
- No. of vessels per control: 6 replicates.
- No. of vessels per abiotic control: 1 or 2 per concentration.

GROWTH MEDIUM
- Standard medium used: Yes M2, the composition of which can be seen in Table 1.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Stock culture medium, M1, and pre-culture medium M2 compositions can be seen in Table 1. Both were prepared using tap water purified by reverse osmosis.

OTHER TEST CONDITIONS
- Photoperiod: Continuously illumination.
- Light intensity and quality: Cool white light of 30 Watt, with an intensity of 62 to 71 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter a spectrophotometer with immersion probe (pathlength =20 mm was used. Cell densities were determined by spectrophotometric measurement of samples at 720 nm. Algal medium was used as a blank and the extra replicates as background for the treated solutions.
- Observation time points: 0, 24, 48 and 72 hours.
- Other:
pH: Measured at the beginning and end of exposure.
Temperature of medium: Measured continuously in a temperature control vessel.
Appearance of cells: At the end of exposure microscopic observations were performed on the highest test concentration to observe for any abnormal appearance in the algae.

TEST CONCENTRATIONS
- The study was performed as a limit test combined with a range-finding test.
- Range finding study
- Test concentrations: Nominal concentrations of 0.10, 1.0 and 10 mg/L.
- Replications: All concentrations were performed in triplicate, one extra replicate was made for each test concentration for sampling after 24 hours.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Growth rate and biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Growth rate and biomass
Details on results:
- No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group.
- Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
- Concentration analysis:
Measured concentrations were show to be stable and in agreement with the nominal, 97 to 99%. The mean recovery of the procedural recovery samples at the concentration of 100 mg/L fell within the criterion of 70-110%. Results are presented in Table 4.
Results with reference substance (positive control):
Algae were exposed for a period of 72 hours to potassium dichromate at concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and to a control. The initial cell density was 1.0 x 104 cells/mL.

Potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/l and higher. The EC50 for growth rate reduction (ERC50: 0-72h) was 2.3 mg/L with a 95% confidence interval ranging from 1.9 to 2.9 mg/L. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/L. Hence, the ERC50: 0-72h for the algal culture tested corresponds with this range. The EC50 for yield inhibition (EYC50: 0-72h) was 0.90 mg/L with a 95% confidence interval ranging from 0.68 to 1.2 mg/L. The historical ranges of the 72h EC50 for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the EYC50: 0-72h for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
CALIBRATION CURVE
Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.

COMPARISON OF AVERAGE GROWTH RATES
The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments:

µi-j = [(lnXj - lnXi) / (tj - ti)] (day^-1)

Where:
µi-j = the average specific growth rate from time i to j
Xi = the biomass at time i
Xj = the biomass at time j

The average growth rate at each test materaial concentration is then compared with the control value and the percentage reduction in growth rate is calculated:

%Ir = [(µC - µT) / µC] x 100

Where:
%Ir = percent inhibition in average specific growth rate
µC = mean value for average specific growth rate in the control group
µT = average specific growth rate for the treatment replicate

YIELD
The percent inhibition in yield is calculated for each treatment replicate as follows:

%Iy = [(YC -YT) / YC] x 100

Where:
%Iy = percent inhibition of yield
YC = mean value for yield in the control group
YT = value for yield for the treatment replicate

DETERMINATION OF THE NOEC AND CALCULATION OF THE EC50
Statistical analysis of the data was not needed as the effects recorded were not biologically significant (<10%).
No EC50 values could be calculated because the test material proved to be non-toxic (EC50 > maximum concentration tested).

Table 1. Mean Cell Densities (x 104cells/mL)

 

Concentration (mg/L)

Exposure Time (hours)

0

24

48

72

Control

1.0

5.3

19.5

61.6

0.10

1.0

5.8

18.9

65.0

1.0

1.0

5.7

17.7

54.7

10

1.0

5.7

19.4

62.2

100

1.0

6.0

19.0

61.0

 

Table 2. Percentage Reduction of Growth Rate and Percentage Inhibition of Yield

 

Concentration (mg/L)

Mean Growth Rate

Yield (0 – 72 hrs)

µ (0 - 72 hrs)

Reduction (%)

x 104cells/mL

Inhibition (%)

Control

0.05718

 

60.60

 

0.10

0.05796

-1.4

64.01

-5.6

1.0

0.05556

2.8

53.70

11.4

10

0.05732

-0.2

61.16

-0.9

100

0.05697

0.4

60.03

0.9

 

Table 3. Percentage Reduction of Growth Rate at Different Time Intervals

 

Concentration (mg/L)

Mean Growth Rate

µ (0 - 24 hrs)

Reduction (%)

µ (24 - 48 hrs)

Reduction (%)

µ (48 - 72 hrs)

Reduction (%)

Control

0.06974

 

0.05391

 

0.04790

 

0.10

0.07312

-4.9

0.04921

8.7

0.05154

-7.6

1.0

0.07250

-4.0

0.04714

12.6

0.04703

1.8

10

0.07277

-4.4

0.05066

6.0

0.04853

-1.3

100

0.07465

-7.0

0.04794

11.1

0.04832

-0.9

 

Table 4. Concentration Analysis

 

Time of Sampling (hours)

Concentration

Relative to nominal (%)

Relative to initial (%)

Nominal (mg/L)

Analysed (mg/L)

0

0

n.d

n.a

 

100

97.3

97

 

1002

99.5

100

 

24

0

n.d

n.a

 

100

97.7

98

100

1002

99.7

100

100

72

0

n.d

n.a

 

100

98.8

99

102

1002

91.9

92

92

2 - Without algae,

n.d – Not detected,

n.a – Not applicable.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, no reduction of growth rate or inhibition of yield was recorded at any of the concentrations tested. The EC50 for both growth rate reduction (ERC50: 0-72h) and yield inhibition (EYC50: 0-72h) exceeded an analytically confirmed nominal concentration of 100 mg/L. The NOEC for both growth rate reduction and yield inhibition was 100 mg/L.
Executive summary:

The toxicity to aquatic algae was determined in a 72 hour static freshwater toxicity study performed using Pseudokirchnerella subcapitata. The study was performed in line with GLP and according to the standardised guidelines OECD 201 and EU Method C.3.

The test organisms were exposed to the test material at the following nominal concentrations; 0.10, 1.0, 10 and 100 mg/L, with a concurrent untreated control. The growth rate and biomass of each sample was observed at 0, 24, 48 and 72 hours. Analysis of samples taken during the test from the nominal 100 mg/L group showed that measured concentrations were stable and in agreement with naminal values, 97 to 99%. Potassium dichromate was run as a positive control, results of whish were in agreement with historical data.

 

Under the conditions of the test, no reduction of growth rate or inhibition of yield was recorded at any of the concentrations tested. The EC₅₀ for both growth rate reduction (ERC₅₀: 0-72h) and yield inhibition (EYC₅₀: 0-72h) exceeded an analytically confirmed nominal concentration of 100 mg/L. The NOEC for both growth rate reduction and yield inhibition was 100 mg/L.

Description of key information

The toxicity of 3,5-Dimethylpyrazole to aquatic algae has been determined to be EC50 > 100 mg/L and NOEC 100 mg/L in a key study (Migchielsen, 2012b) performed according to OECD 201 and EU Method C.3.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

Migchielsen (2012b) has been provided as the key study where the toxicity of the test material to aquatic algae was determined to be EC₅₀> 100 mg/L with a NOEC of 100 mg/L. The study was performed to GLP and standardised guidelines and had thus been assigned a reliability score of 1 using the principles for assessing data quality as set out in Klimisch (1997).

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