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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:
04 March 2016 to 15 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: At the start of the test (0 hours), ca 20 mL samples of freshly prepared test media were taken from the control, 1.0, 3.2, 10, 32 and 100 % saturated solution test media preparation flasks for chemical analysis.
At 72 hours, replicate test vessels at each treatment level were pooled and sampled. Samples (ca 20 mL) of each were taken for chemical analysis.
At each sampling occasion, duplicate samples were taken. One for immediate chemical analysis and one as a "back up" sample should further analysis be required.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: At the start of the definitive test, the 100 % saturated solution test concentration was prepared by weighing ca 100 mg of test substance and adding to ca 1 000 mL of EC medium. This was then stirred for ca 24 hours and filtered through 0.45 µm syringe filters. A serial dilution was then performed from the resulting test media to give the remaining test concentrations, after which it was observed to be a colourless solution. A control treatment was prepared by adding EC medium only to the control vessels.
- Controls: Six test vessels were prepared for the control vessels (algal nutrient medium only) and three test vessels were prepared for each of the five test concentrations, each with an initial cell concentration of 1 × 10^4 cells/mL.
- Concentration of vehicle in test medium: Based on the results of the range-finding test, the definitive test was conducted with a control and nominal test concentrations of 1.0, 3.2, 10, 32 and 100 % saturated solution (0.764, 2.29, 5.24, 22.6 and 59.6 mg/L based on geometric mean measured concentrations).
- Evidence of undissolved material: The appearance, colour and behaviour of the test substance in the test media were recorded at the start and end of the test. At 0 hours, the control and 1.0, 3.2, 10, 32 and 100 % saturated solution test groups were observed as being colourless solutions. At the end of the test (72 hours), the control and the 1.0, 3.2, 10, 32 and 100 % saturated solution test groups were observed as pale green homogeneous hazy dispersions of algal cells.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: Strain 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Oban, UK. On receipt from the CCAP, the slope culture was stored in the fridge at 2-8 °C. A typical shelf life for each slope was 8 months, after which the slopes were discarded.
- Age of inoculum: Prior to each test, a sub-sample (ca 200 µL) of a current liquid slope culture was added to two starter cultures (conical flasks) containing 100 mL EC medium.
Each culture was incubated (under the same conditions as used in the test) for at least 72 hours prior to the start of each test. Following incubation, the cell density of a single starter culture was established using a particle counter (Z2 Coulter Counter®) and confirmed manually using a haemocytometer.
- Method of cultivation: Semi-axenic cultures of Pseudokirchneriella subcapitata were maintained in liquid culture. These were used to inoculate starter liquid cultures, which in turn were used to inoculate test vessels containing control and test media at the start of each test.
To prepare the alga culture medium, stock solutions containing the various nutrients were prepared with reverse osmosis (RO) water. Aliquots of each of the stock solutions were then added to RO water. The alga culture medium was autoclaved. Once cooled, an aliquot of a stock solution of NaHCO3 was added to the alga culture medium through a sterile filter (0.2 µm filter pore size).

ACCLIMATION
- Culturing media and conditions: Prior to testing, duplicate starter cultures were prepared and incubated under test conditions to obtain sufficient algal cells in exponential growth and to achieve a starting algae cell density of 1 × 10^4 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.1 – 22.6 °C
pH:
6.50 to 7.45
Nominal and measured concentrations:
Definitive test: Nominal test concentrations of 1.0, 3.2, 10, 32 and 100 % saturated solution (0.764, 2.29, 5.24, 22.6 and 59.6 mg/L based on geometric mean measured concentrations).
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile autoclaved glass 250 mL Erlenmeyer (conical) flasks.
- Type: Closed. All flasks were loosely-capped.
- Aeration: All vessels were loosely capped and placed on an orbital shaker (ca 100 rpm).
- Material, size, headspace, fill volume: 100 mL of the appropriate control or test media was added.
- No. of organisms per vessel: Algal cell concentration of 1 × 10^4 cells/mL.
- No. of vessels per concentration: Three test vessels were prepared for each of the five test concentrations.
- No. of vessels per control: Six test vessels.
- Control: A media blank (EC Medium only) was prepared to establish background counts on each sampling occasion. Background counts were subtracted from the cell containing results for each of the inoculated test vessels. The resulting cell counts were then used to calculate the area under the growth curves, yield and the corresponding specific growth rates.

GROWTH MEDIUM
- Standard medium used: EC medium
- Detailed composition if non-standard medium was used: See Preparation of Algal Medium table, below.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No At the start of the test, the pH of freshly prepared test media was determined. The pH of each vessel was also determined at the end of the test.
- Photoperiod: 72 hours continuous light.
- Light intensity and quality: 4440 - 8880 Lux light intensity using LED lights, emitting light across the visible portion of the spectrum (400 - 700 nm). The light intensity within the test area was monitored at the start and end of the test.
- Temperature: The incubator temperature was set within the range 21 to 24 °C and maintained within ± 2 °C for the duration of the test. The temperature was recorded continuously using a digital temperature logger.

EFFECT PARAMETERS MEASURED:
- Test vessel sampling: At approximately 24-hour intervals after the start of the incubation period, pre-determined volumes of test media (1.0 mL at 24 hours and 0.5 mL at 48, and 72 hours) were removed from each incubated test vessel, and transferred to individually identified cell counting vials. The contents of each vial were diluted to a 10 mL final volume with an electrolyte solution. The cell density of the vial contents was then determined using a particle counter (Z2 Coulter Counter®).

TEST CONCENTRATIONS
- Range finding study: Yes.
Duplicate test vessels were prepared for the control and each test concentration with an initial algae cell concentration of 1 × 10^4 cells/mL. Based on nominal concentrations, the results of the range-finding test suggested that the 72-hour ExC50 values would be greater than 100 % saturated solution.
- Test concentrations: Nominal test substance concentrations of 0.1, 1.0, 10 and 100 % saturated solutions.
- Results used to determine the conditions for the definitive study: Yes.
At the start of the test, the control and each test concentration were observed to be colourless solutions. At 72 hours, the control and the 0.1, 1.0, and 10 % saturated solution test concentrations were observed to be green, homogenous hazy dispersions of algae cells; the 100 % saturated solution test concentration was observed to be light green homogenous hazy dispersions of algal cells. Analysis of the test concentrations at 0 hours showed measured concentrations to range from 0.0843 to 55.2 mg/L. Analysis of the test concentrations after 72 hours showed measured concentrations to range from 0.0616 to 50.2 mg/L. These results indicated that the test substance was stable in the test media over the 72-hour period. Analysis of prepared non-inoculated test media after 72 hours indicated that adsorption to algae was not likely to be an issue. The results of the range-finding test suggested that the 72-hour EC50 values for yield, specific growth rate and area under the curve (biomass) were likely to be greater than concentration of the 100 % saturated solution, based on nominal test substance concentration.
Based on the results of a range-finding test, for which only the key findings have been reported, the definitive test was conducted at nominal test concentrations of 1.0, 3.2, 10, 32 and 100 % saturated solutions (0.764, 2.29, 5.24, 22.6 and 59.6 mg/L based on geometric mean measured concentrations).
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 59.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate, cell number and biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
59.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Any observations that might cause a difference between measured and nominal values: The freshly prepared test media at 0 hours appeared as colourless solutions. Test media at the end of the definitive test was pale green in colour and described as a homogenous hazy dispersion of algal cells.

- Any stimulation of growth found in any treatment: Based on geometric mean measured concentrations, the 72-hour EyC50, EbC50 and ErC50 values were calculated to be >59.6 mg/L. The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 59.6 mg/L.
Reported statistics and error estimates:
Statistical analysis was performed using the CETIS program v 1.8.6.8.
To distinguish between ExC50 values, estimated using areas under the growth curve, final yield and growth rates, the symbols EbC50, EyC50 and ErC50 were used, respectively.
The EC10, EC20 and EC50 are defined as the concentrations that result in a 10 %, 20 % and 50 % mean reduction, respectively, relative to the control.
The area under the growth curve (A), the average specific growth rate (μ) and the final yield over specified time intervals were analysed using Parametric-Multiple Comparison test to determine the no observed effect concentration (NOEC).
Linear interpolation analysis was performed in order to estimate EC10, EC20 and EC50 values. Where possible, 95 % confidence limits were calculated for the EC10, EC20 and EC50 values.

Chemical Analysis

Analysis of the freshly prepared test solutions at 0 hours showed measured concentrations of 0.694, 2.18, 7.84, 22.4 and 81.4 mg/L.

Analysis of the 72-hour test solutions showed measured concentrations of 0.840, 2.41, 3.50, 22.9, 43.7 mg/L. The results showed some variability concentration over the 72- hour test period, 2 concentrations showed a 50 % decline in concentration, two concentrations showed no significant decline and one concentration had an increase in concentration. Given this variability, it was considered justifiable to base the results on geometric mean measured concentrations. These were calculated to be 0.764, 2.29, 5.24, 22.6, and 59.6 mg/L.

Environmental Conditions

Temperatures remained within the 21 - 24 ºC, and were maintained within the ± 2 ºC, range established for the test. The light intensity was measured at the start and end of the test; 7840 Lux at the start and 7920 Lux at the end of the test.

The pH of the test solutions ranged from 6.50 to 7.45 at test initiation. The pH tended to increase relative to increases in algal densities, which is typical for tests conducted with P. subcapitata. The pH in the control increased by a maximum of 3.0 units, which exceeds the OECD 201 guideline requirement of an increase, not greater than 1.5 units. However, an elevated pH value is an indicator of good algal cell growth, whereby the release of oxygen from the algal cells during normal photosynthetic activity promotes an alkaline pH in the surrounding test media. This change in pH was not considered to be detrimental to this study since increases in pH of greater than 1.5 units are commonly observed and this change had no impact on the growth of the control, which met the established validity criteria for the test.

Validity Criteria

Parameter

Criterion

Observed values
Control

Control Cell Density Increase

Increase by a factor of at least 16

83

Coefficient of variation
of average specific growth rates at 72h

≤ 7 %

3.17

Mean Coefficient of Variation
for Section by Section Specific Growth Rates
(All 6 Replicates – 0-24, 24-48, 48-72 hours)

≤ 35 %

7.82

Mean Coefficient of Variation
for Section by Section Specific Growth Rates
(Individual Replicates – 0-24, 24-48, 48-72, 0-48,
0-72, 24‑72 hours)

≤ 35 %

7.09

Mean Coefficient of Variation
for Section by Section Specific Growth Rates
(Individual Replicates – 0-24, 24-48, 48-72 hours)

≤ 35 %

10.57

All validity criteria were met, therefore the test was considered valid.

Validity criteria fulfilled:
yes
Conclusions:
Based on geometric mean measured concentrations, the 72-hour EyC50, EbC50 and ErC50 values were calculated to be >59.6 mg/L. The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 59.6 mg/L under the experimental conditions of the study.
Executive summary:

The acute toxicity of the test material to the freshwater alga was investigated in a study performed under GLP conditions in accordance to the standardised guideline OECD 201.

The test was conducted on the green alga, Pseudokirchneriella subcapitata at the exponential growth phase. Based on the results of a range-finding test, the definitive test was conducted at nominal test concentrations of 1.0, 3.2, 10, 32 and 100 % saturated solutions (0.764, 2.29, 5.24, 22.6 and 59.6 mg/L based on geometric mean measured concentrations).

Test vessels (250 mL conical flasks) were prepared containing 100 mL of the appropriate test or control medium. Each test vessel was inoculated with 1 × 10^4 algae cells/mL and incubated at 21 - 24 °C (under 4440 - 8880 Lux light intensity) for 72 hours with cell counts at 24-hour intervals.

Analysis of the test media samples was conducted at 0 and 72 hours.

Measured test concentrations were determined from samples of test solution from each treatment and control group at the beginning and end of the test. All validity criteria were met, therefore the test was considered valid. The results showed some variability concentration over the 72-hour test period, 2 concentrations showed a 50 % decline in concentration, 2 concentrations showed no significant decline and one concentration had an increase in concentration. Given this variability, it was considered justifiable to base the results on geometric mean measured concentrations. These were calculated to be 0.764, 2.29, 5.24, 22.6, and 59.6 mg/L.

Analysis of the test samples at 0 hours showed measured concentrations to range from 0.694 to 81.4 mg/L. Samples at 72 hours showed measured concentrations to range from 0.840 to 43.7 mg/L.

Based on geometric mean measured concentrations, the 72-hour EyC50, EbC50 and ErC50 values were calculated to be >59.6 mg/L. The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 59.6 mg/L under the experimental conditions of the study.

Description of key information

Based on geometric mean measured concentrations, the 72-hour EyC50, EbC50 and ErC50 values were calculated to be >59.6 mg/L. The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 59.6 mg/L under the experimental conditions of the study.

Key value for chemical safety assessment

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

Additional information

The acute toxicity of the test material to the freshwater alga was investigated in a study performed under GLP conditions in accordance to the standardised guideline OECD 201. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The test was conducted on the green alga, Pseudokirchneriella subcapitata at the exponential growth phase.Based on the results of a range-finding test, the definitive test was conducted at nominal test concentrations of 1.0, 3.2, 10, 32 and 100 % saturated solutions (0.764, 2.29, 5.24, 22.6 and 59.6 mg/L based on geometric mean measured concentrations).

Test vessels (250 mL conical flasks) were prepared containing 100 mL of the appropriate test or control medium. Each test vessel was inoculated with 1 × 10^4 algae cells/mL and incubated at 21 - 24 °C (under 4440 - 8880 Lux light intensity) for 72 hours with cell counts at 24-hour intervals.

Analysis of the test media samples was conducted at 0 and 72 hours.

Measured test concentrations were determined from samples of test solution from each treatment and control group at the beginning and end of the test. All validity criteria were met, therefore the test was considered valid. The results showed some variability concentration over the 72-hour test period, 2 concentrations showed a 50 % decline in concentration, 2 concentrations showed no significant decline and one concentration had an increase in concentration. Given this variability, it was considered justifiable to base the results on geometric mean measured concentrations. These were calculated to be 0.764, 2.29, 5.24, 22.6, and 59.6 mg/L.

Analysis of the test samples at 0 hours showed measured concentrations to range from 0.694 to 81.4 mg/L. Samples at 72 hours showed measured concentrations to range from 0.840 to 43.7 mg/L.

Based on geometric mean measured concentrations, the 72-hour EyC50, EbC50 and ErC50 values were calculated to be >59.6 mg/L. The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 59.6 mg/L under the experimental conditions of the study.