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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
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
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Analytical measurement was performed at the applied test concentration levels at the beginning of the test and at 24 hour intervals thereafter during the experiment. Samples from the control were taken for analysis at the start and at the end of the test only. The lowest test substance concentration (0.3 mg/L, nominal) was not measured as it was below the detection limit of the method.
The samples were analysed by GC with FID detection method.
Vehicle:
no
Details on test solutions:
Formulation of the test substance:

A stock solution with a concentration of 100.0 mg/L (nominal) was prepared with direct addition of the test substance, mixed into the test medium (OECD Medium) using ultrasonic bath (~15 minutes). The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae. The nominal concentrations in the main experiment were: 0.3, 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L.

Untreated Control:

Algal growth medium was inoculated with algal cells (without test substance) and was examined in parallel to the test substance concentrations.




Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Culture conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was 22.7 °C measured in the flask and between 21.4 and 23.3 °C measured within the climate chamber.
pH:
The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.64 – 9.06 during the experiment.
Nominal and measured concentrations:
The nominal concentrations in the main experiment: 0.3, 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L
The measured and calculated geometric mean test substance concentrations: 0.29, 0.92, 2.72, 7.61, 27.06 and 93.10 mg/L
Details on test conditions:
Preliminary Range Finding Test

Algal cells were exposed to each concentration of the test substance plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group.

Main Experiment:

Because significant inhibition was observed at the examined concentration levels during the preliminary range-finding test, six test concentrations in a geometric series with a separation factor of 3.2 and one control were tested in the main experiment.

The exposure time was 72 hours. The test was started (0 hours) by inoculation of a biomass of approximately 10+E4 algal cells per mL test medium.

The test was performed with five replicates per test concentration (with the exception of the lowest test level); three replicates were used for observations and one - one replicate was used for the 24 and 48 hours analytical measurements. Six replicates were used in the control group. Volumes of 100 mL algal suspension per replicate in 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.

Observations:

The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
Microscopic observation of the algal cells in each concentration and in the control was performed (at 24 h, 48 h and 72 h) to detect any abnormal appearance of the algae.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.71 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. limits: 8.49 – 11.11
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.72 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.07 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95 % conf. limits: 4.48 – 5.73
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.92 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.54 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % conf. limits: 4.84 – 6.34
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.92 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
With respect to the inhibitory effect of the test substance, the 0-72 h average specific growth rates were significantly different from that of the control group in the examined concentration range of 7.61 – 93.10 mg/L (measured), therefore the NOEC was determined as 2.72 mg/L (measured) and the LOEC was determined as 7.61 mg/L (measured). The 0-72 h areas and yield were significantly different from that of the control group in the examined concentration range of 2.72 – 93.10 mg/L (measured). The NOEC was determined as 0.92 mg/L (measured); the LOEC was determined as 2.72 mg/L (measured).

Validity criteria:
- The cell density in the control cultures increased by the factor of 69.67 within three days.
- The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 11.13 %.
- The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 1.02 %.
- All validity criteria were met, therefore the study can be considered as valid.
Results with reference substance (positive control):
For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 17/058-022AL) with the reference substance Potassium dichromate is (Batch Number: A0345704): 07 – 10 March 2017.
The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EyC 50: 0.54 mg/L, (95 % confidence limits: 0.50 – 0.59 mg/L)
The 72h EbC 50: 0.64 mg/L, (95 % confidence limits: 0.59 – 0.70 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).
Reported statistics and error estimates:
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel 2007 for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The ErC50, EbC50 and EyC50 values of the test substance and their confidence limits were calculated using Probit analysis by TOXSTAT software.
Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test ( = 0.05) by TOXSTAT software.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

Growth Rates (m) and Percentage Inhibition ofmduring the Test Period

Concentration

Growth rate (m) and % inhibition ofm

Nominal

Measured

0–24 h

0–48 h

0–72 h

[mg/L]

[mg/L]

m

%

m

%

m

%

Control

0.0

0.0538

0.0

0.0596

0.0

0.0589

0.0

0.3

0.29

0.0538

0.0

0.0594

0.3

0.0589

0.0

1.0

0.92

0.0538

0.0

0.0586

1.7

0.0586

0.6

3.1

2.72

0.0538

0.0

0.0582

2.4

0.0572

2.9

9.8

7.61

0.0345*

35.8

0.0376*

36.9

0.0403*

31.7

31.3

27.06

0.0096*

82.1

0.0048*

91.9

0.0032*

94.6

100.0

93.10

0.0000*

100.0

0.0000*

100.0

0.0000*

100.0

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Concentration

Area under the Growth Curves (A) and Percentage Inhibition of A

Nominal

Measured

0–24 h

0–48 h

0–72 h

[mg/L]

[mg/L]

A

%

A

%

A

%

Control

0.0

32.0

0.0

262.0

0.0

1284.0

0.0

0.3

0.29

32.0

0.0

260.0

0.8

1280.0

0.3

1.0

0.92

32.0

0.0

252.0

3.8

1244.0

3.1

3.1

2.72

32.0

0.0

248.0

5.3

1160.0*

9.7

9.8

7.61

16.0*

50.0

96.0*

63.4

368.0*

71.3

31.3

27.06

4.0*

87.5

12.0*

95.4

20.0*

98.4

100.0

93.10

0.0*

100.0

0.0*

100.0

0.0*

100.0

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

Yield (Y) and Percentage Inhibition of Y during the Test Period

Concentration

Concentration

Nominal

Measured

0–72 h

[mg/L]

[mg/L]

Y

%

Control

0.0

68.7

0.0

0.3

0.29

68.7

0.0

1.0

0.92

67.0

2.4

3.1

2.72

60.7*

11.7

9.8

7.61

17.3*

74.8

31.3

27.06

0.3*

99.5

100.0

93.10

0.0*

100.0

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the 72 h NOEC for the growth rate was determined as 2.72 mg/L (measured) and the 72 h EC50 was determined as 9.71 mg/L (measured). The 72 h NOEC for the biomass was determined as 0.92 mg/L (measured) and the 72 h EC50 was determined as 5.54 mg/L (measured).
Executive summary:

A study was conducted to determine the effect of the test substance on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 h according to OECD Guideline 201, in compliance with GLP. As significant inhibition was observed during the preliminary range-finding test, six test concentrations in a geometric series (factor 3.2) and one control were tested in the main experiment. The nominal concentrations in the main experiment were: 0.3, 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L. Test concentrations were analytically determined at the start of the experiment and at 24 hour intervals thereafter. The lowest test substance concentration (0.3 mg/L, nominal) was not measured as it was below the limit of detection of the method. The biological results are based on the measured and calculated geometric mean concentrations. The corresponding measured and calculated geometric mean test substance concentrations were: 0.29, 0.92, 2.72, 7.61, 27.06 and 93.10 mg/L. The test design included five replicates at each test concentration (with the exception of the lowest test level) and six replicates for the untreated controls. Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software. The ErC50, EbC50 and EyC50 values of the test substance and their confidence limits were calculated using Probit analysis by TOXSTAT software. Under the study conditions, the 72 h NOEC for the growth rate was determined as 2.72 mg/L (measured) and the 72 h EC50 was determined as 9.71 mg/L (measured). The 72 h NOEC for the biomass was determined as 0.92 mg/L (measured) and the 72 h EC50 was determined as 5.54 mg/L (measured) (Sipos, 2017).

Description of key information

Key value for chemical safety assessment

EC50 for freshwater algae:
9.71 mg/L
EC10 or NOEC for freshwater algae:
2.72 mg/L

Additional information

A study was conducted to determine the effect of the test substance on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 h according to OECD Guideline 201, in compliance with GLP. As significant inhibition was observed during the preliminary range-finding test, six test concentrations in a geometric series (factor 3.2) and one control were tested in the main experiment. The nominal concentrations in the main experiment were: 0.3, 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L. Test concentrations were analytically determined at the start of the experiment and at 24 hour intervals thereafter. The lowest test substance concentration (0.3 mg/L, nominal) was not measured as it was below the limit of detection of the method. The biological results are based on the measured and calculated geometric mean concentrations. The corresponding measured and calculated geometric mean test substance concentrations were: 0.29, 0.92, 2.72, 7.61, 27.06 and 93.10 mg/L. The test design included five replicates at each test concentration (with the exception of the lowest test level) and six replicates for the untreated controls. Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software. The ErC50, EbC50 and EyC50 values of the test substance and their confidence limits were calculated using Probit analysis by TOXSTAT software. Under the study conditions, the 72 h NOEC for the growth rate was determined as 2.72 mg/L (measured) and the 72 h EC50 was determined as 9.71 mg/L (measured). The 72 h NOEC for the biomass was determined as 0.92 mg/L (measured) and the 72 h EC50 was determined as 5.54 mg/L (measured) (Sipos, 2017).