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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:
20 March 2018 to 23 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
see "Any other information on materials and methods incl. tables"
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
yes
Remarks:
see "Any other information on materials and methods incl. tables"
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
see "Any other information on materials and methods incl. tables"
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
The analytical phase of this study was conducted at the Test Site, Seibersdorf LaborGmbH, in Austria.
The concentration of Cadmium, as a marker for the test item was measured at all tested concentration levels at the beginning and at the end of the experiment using a
suitable ICP method.

Samples were taken from the control (three replicates) and at the applied test concentration levels (each replicate) at the beginning and at the end of the experiment.
Duplicate samples were taken in plastic tubes, acidified with pure nitric acid (9 mL sample + 1 mL HNO3) (stored under test conditions until analysis) in agreement with the requirements of the Principal Investigator. To avoid any problems with micro particles, therefore the samples were membrane filtrated before acidification.

After sampling, one set of the samples was stored under test conditions and then sent to the Test Site for analysis. One set of the samples is retained as a back-up at the Test Facility, thus these samples were frozen and kept approximately at -20°C at the Test Facility, if required for any confirmatory analyses (discarded after satisfactory results were obtained on the first set of).
Vehicle:
yes
Details on test solutions:
Because the test item is very poorly soluble in water, a test solution was prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.

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

Reference 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: 17/367-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 04 – 07 December 2017.
The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EbC 50: 0.63 mg/L, (95 % confidence limits: 0.58 – 0.69 mg/L)
The 72h EyC 50: 0.53 mg/L, (95 % confidence limits: 0.49 – 0.58 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Dilution Water
Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.

A saturated test item solution (nominal loading rate of 100 mg/L) was prepared by dispersing/dissolving the amount of test item into the test medium (OECD Medium) two
days before the start of the study. This solution was shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The
non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give the 100 % saturated solution.

The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)

Source: The algae were supplied by the SAG: Collection of AlgalCultures, 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:
other: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
Not specified
Post exposure observation period:
Not specified
Hardness:
Not specified
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 between 22.6 and 22.9 °C measured in the flask and between 21.0 and 23.2 °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 7.16 – 8.22 during the experiment.
Dissolved oxygen:
Not specified
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
The following nominal concentrations were tested: 1.0, 3.1, 9.8, 31.3 and 100.0% saturated solutions.

The corresponding measured geometric mean test item concentrations were: 5.63, 16.37, 54.24, 181.97 and 597.22 μg/L (based on the calculated Cadmium content of 58.737% in the test item).
Details on test conditions:
DESCRIPTION OF THE TEST PROCEDURE
The exposure time was 72 hours. The test was started (0 hours) by inoculation of a biomass of approximately 104 algal cells per mL test medium.
The test was performed with three replicates per test concentration and six replicates 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.

Preliminary Range Finding Test
A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group. During the formulation procedure was prepared by the similar method described above (3.1.2.).

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 24h, 48h and 72h) to detect any abnormal appearance of the algae.

DEVIATION FROM THE STUDY PLAN
Concerning: Test conditions, light intensity
According to the Study Plan: The differences in light intensity between the test vessels should not exceed 15 % variation.
Deviation: The difference in light intensity between the test vessels was 17.2 % in one case (from ten).
Reason for this change: Technical.
Presumed Effect on the Study: None. Due to that all of the validity criteria were met in the untreated control, the study can be considered as valid.
Light intensity
The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 7813 lux (equivalent to ~106 μE/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400 -700 nm). The differences in light intensity between the test vessels were within ± 15% (with the exception of only one case where the difference was 17.2 %) and therefore provided equal conditions for each test vessel.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
84.47 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
Cadmium content of 58.737 % in the test item
Basis for effect:
growth rate
Remarks on result:
other: 95% C.I 73.92 – 96.53
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
40.69 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95% C.I 36.05 – 45.92
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
43.56 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% C.I 38.27 – 49.58
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
16.37 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
16.37 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
16.37 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
54.24 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
54.24 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
54.24 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
VALIDITY
The cell density in the control cultures increased by the factor of 70.17 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 10.50 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.78 %. All validity criteria were met, therefore the study can be considered as valid.

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS
Chuff cells were observed at the nominal concentration of 9.8 % saturated solution (nominal) at the end of the test and in the two highest examined concentrations of 31.3 and 100.0 % saturated solutions (nominal) during the whole experiment.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
see "Any other information on results incl. tables"

AVERAGE SPECIFIC GROWTH RATES

The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value at the nominal concentration range of 9.8 - 100.0 % saturated solution (corresponding to 54.24 - 597.22 μg/L, measured), accordingly the No Observed Effect Concentration (NOEC) was determined as 3.1 % saturated solution (nominal) (corresponding to 16.37 μg/L, measured). The 72 h ErC50 value was determined [by Probit analysis (TOXSTAT software)] as 14.99 % saturated solution (95 % confidence limits: 13.19 – 17.05 % saturated solution), calculation based on the nominal concentrations. The 72 h ErC50 value was determined [by Probit analysis (TOXSTAT software)] as 84.47 μg/L (95 % confidence limits: 73.92 – 96.53 μg/L), calculation based on the measured concentrations.

AREAS UNDER THE GROWTH CURVES

The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h areas were statistically significantly different from the untreated control value at the nominal concentration range of 9.8 - 100.0 % saturated solution (corresponding to 54.24 - 597.22 μg/L, measured), accordingly the No Observed Effect Concentration (NOEC) was determined as 3.1 % saturated solution (nominal) (corresponding to 16.37 μg/L, measured).

The 72 h EbC50 value was determined [by Probit analysis (TOXSTAT software)] as 7.93% saturated solution (95 % confidence limits: 7.00 – 8.98 % saturated solution), calculation based on the nominal concentrations. The 72 h EbC50 value was determined [by Probit analysis (TOXSTAT software)] as 43.56 μg/L (95 % confidence limits: 38.27 – 49.58 μg/L), calculation based on the measured concentrations.

YIELD

The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h yield was statistically significantly different from the untreated control value at the nominal concentration range of 9.8 - 100.0 % saturated solution (corresponding to 54.24 - 597.22 μg/L, measured), accordingly the No Observed Effect Concentration (NOEC) was determined as 3.1 % saturated solution (nominal) (corresponding to 16.37 μg/L, measured).

The 72 h EyC50 value was determined [by Probit analysis (TOXSTAT software)] as 7.42% saturated solution (95 % confidence limits: 6.60 – 8.34 % saturated solution), calculation based on the nominal concentrations. The 72 h EbC50 value was determined [by Probit analysis (TOXSTAT software)] as

40.69 μg/L (95 % confidence limits: 36.05 – 45.92 μg/L), calculation based on the measured concentrations.

Validity criteria fulfilled:
yes
Conclusions:
The effect of Cadmium selenide test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), over an exposure period of 72 hours. With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group at the examined nominal concentration range of 9.8 – 100.0 % saturated solution (corresponding to 54.24 – 597.22 μg/L, measured), therefore the NOEC was determined as 3.1 % saturated solution (nominal) (16.37 μg/L, measured) and LOEC was determined as 9.8 % saturated solution (nominal) (54.24 μg/L, measured)
Executive summary:

The effect of test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

As significant toxic response was observed at the examined concentration levels during the preliminary range-finding test, five test concentrations in a geometric series (factor 3.2) and one control were tested in the main experiment.

The nominal concentrations of test item used in the main experiment were: 1.0, 3.1, 9.8, 31.3 and 100.0 % saturated solutions.

Test concentrations were analytically determined at the start and at the end of the experiment by measuring the concentration of Cadmium (as a marker for the test item) using a suitable ICP method. The corresponding measured geometric mean test item concentrations were: 5.63, 16.37, 54.24, 181.97 and 597.22 μg/L (based on the calculated Cadmium content of 58.737 % in the test item).

The biological results are based on both the nominal percentage saturated solutions and measured geometric mean test item concentrations.

The test design included three replicates at each test concentration 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 item and their confidence limits were calculated using Probit analysis by TOXSTAT software. With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group at the examined nominal concentration range of 9.8 – 100.0 % saturated solution (corresponding to 54.24 – 597.22 μg/L, measured), therefore the NOEC was determined as 3.1 % saturated solution (nominal) (16.37 μg/L, measured) and LOEC was determined as 9.8 % saturated solution (nominal) (54.24 μg/L, measured). The biological results are summarised in the following tables:

Influence of Cadmium selenide on the Growth of Pseudokirchneriella subcapitata, calculation based on the nominal concentration (% saturated solution of 100 mg/L nominal loading rate)

Parameter

(0-72 h)

Growth rate (r)

[% sat. sol.]

Yield (y)

[% sat. sol.]

Biomass (b)

[% sat. sol.]

EC50

14.99

7.42

7.93

95 % conf. limits

13.19 – 17.05

6.60 – 8.34

7.00 – 8.98

NOEC

3.1

3.1

3.1

LOEC

9.8

9.8

9.8

Calculation based on the nominal concentrations.

 

Influence of Cadmium selenide on the Growth of Pseudokirchneriella subcapitata, calculation based on the measured test item concentrations

Parameter

(0-72 h)

Growth rate (r)

[μg/L]

Yield (y)

[μg/L]

Biomass (b)

[μg/L]

EC50

84.47

40.69

43.56

95 % conf. limits

73.92 – 96.53

36.05 – 45.92

38.27 – 49.58

NOEC

16.37

16.37

16.37

LOEC

54.24

54.24

54.24

Calculation based on the measured concentrations

Description of key information

The effect of Cadmium selenide test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), over an exposure period of 72 hours. With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group at the examined nominal concentration range of 9.8 – 100.0 % saturated solution (corresponding to 54.24 – 597.22 μg/L, measured), therefore the NOEC was determined as 3.1 % saturated solution (nominal) (16.37 μg/L, measured) and LOEC was determined as 9.8 % saturated solution (nominal) (54.24 μg/L, measured)

Key value for chemical safety assessment

EC50 for freshwater algae:
43.56 µg/L
EC10 or NOEC for freshwater algae:
16.37 µg/L

Additional information

The effect of test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

As significant toxic response was observed at the examined concentration levels during the preliminary range-finding test, five test concentrations in a geometric series (factor 3.2) and one control were tested in the main experiment.

The nominal concentrations of test item used in the main experiment were: 1.0, 3.1, 9.8, 31.3 and 100.0 % saturated solutions.

Test concentrations were analytically determined at the start and at the end of the experiment by measuring the concentration of Cadmium (as a marker for the test item) using a suitable ICP method. The corresponding measured geometric mean test item concentrations were: 5.63, 16.37, 54.24, 181.97 and 597.22 μg/L (based on the calculated Cadmium content of 58.737 % in the test item).

The biological results are based on both the nominal percentage saturated solutions and measured geometric mean test item concentrations.

The test design included three replicates at each test concentration 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 item and their confidence limits were calculated using Probit analysis by TOXSTAT software. With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group at the examined nominal concentration range of 9.8 – 100.0 % saturated solution (corresponding to 54.24 – 597.22 μg/L, measured), therefore the NOEC was determined as 3.1 % saturated solution (nominal) (16.37 μg/L, measured) and LOEC was determined as 9.8 % saturated solution (nominal) (54.24 μg/L, measured). The biological results are summarised in the following tables:

Influence of Cadmium selenide on the Growth of Pseudokirchneriella subcapitata, calculation based on the nominal concentration (% saturated solution of 100 mg/L nominal loading rate)

Parameter

(0-72 h)

Growth rate (r)

[% sat. sol.]

Yield (y)

[% sat. sol.]

Biomass (b)

[% sat. sol.]

EC50

14.99

7.42

7.93

95 % conf. limits

13.19 – 17.05

6.60 – 8.34

7.00 – 8.98

NOEC

3.1

3.1

3.1

LOEC

9.8

9.8

9.8

Calculation based on the nominal concentrations.

 

Influence of Cadmium selenide on the Growth of Pseudokirchneriella subcapitata, calculation based on the measured test item concentrations

Parameter

(0-72 h)

Growth rate (r)

[μg/L]

Yield (y)

[μg/L]

Biomass (b)

[μg/L]

EC50

84.47

40.69

43.56

95 % conf. limits

73.92 – 96.53

36.05 – 45.92

38.27 – 49.58

NOEC

16.37

16.37

16.37

LOEC

54.24

54.24

54.24

Calculation based on the measured concentrations