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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:
15 September 2017 to 06 October 2017
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)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Samples for possible analysis were taken from all test concentrations and the control at t=0 h, t=24 h and t=72 h. 4.8 mL was taken from the approximate centre of the test vessels.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15 °C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Vehicle:
no
Details on test solutions:
- Preparation of test solutions started with a loading rate of 100 mg/L applying a 10 minute treatment with ultrasonic waves followed by a 3 day period of magnetic stirring to ensure maximum dissolution of the test material in medium. Thereafter, the aqueous Saturated Solution (SS) was collected by means of filtration through a 0.45 µm membrane filter and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colourless at the end of the preparation procedure.
- After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
- Any residual volumes were discarded.
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 in a climate room at a temperature of 21-24 °C with light intensity of 60 to 120 µE/m"2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
22-23 °C
pH:
7.7-8.1
Nominal and measured concentrations:
Nominal concentrations: 1.0, 10 and 100 % solutions
Measured concentration at 100 % solution: 13 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL glass vessels, capped.
- Material, size, headspace, fill volume: 50 mL
- During incubation the algal cells were kept in suspension by continuous shaking.
- Initial cells density: 1 x 10^4 cells/mL.
- No. of vessels per concentration (replicates): 6 for the highest concentration (100 % SS), 3 replicates of each intermediate test concentration. 1 or 2 extra replicate of each test concentration for sampling purposes after 24 hours.
- No. of vessels per control (replicates): 6

CULTURE MEDIUM
- Stock culture medium: M1; according to the NPR 6505 with the following composition NaNO3 500 mg/L, K2HPO4.3H2O 52 mg/L, MgSO4.7H2O 75 mg/L, Na2CO3.10H2O 54 mg/L, C6H8O7.H2O 6 mg/L, NH4NO3 330 mg/L, CaCl2.2H2O 35 mg/L, C6H5FeO7.xH2O 6 mg/L, H3BO3 2.9 mg/L, MnCl2.4H2O 1.81 mg/L, ZnCl2 0.11 mg/L, CuSO4.5H2O 0.08 mg/L and (NH4)6Mo7O24.4H2O 0.018 mg/L.
-Pre-culture: 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 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

TEST MEDIUM / WATER PARAMETERS
- M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition: NH4Cl 15 mg/L, MgCl2.6H2O 12 mg/L, CaCl2.2H2O 18 mg/L, MgSO4.7H2O 15 mg/L, KH2PO4 1.6 mg/L, FeCl3.6H2O 64 µg/L, Na2EDTA.2H2O 100 µg/L, H3BO3 185 µg/L, MnCl2.4H2O 415 µg/L, ZnCl2 3 µg/L, CoCl2.6H2O 1.5 µg/L, CuCl2.2H2O 0.01 µg/L, Na2MoO4.2H2O 7 µg/L and NaHCO3 50 mg/L.
- Intervals of water quality measurement: pH was measured at the beginning of the test for the control and the highest test concentration and at the end of the test for all test concentrations. Temperature of medium was continuously monitored in a temperature control vessel.


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 78 to 83 µE.m^-2.s^-1.

EFFECT PARAMETERS MEASURED
- Appearance of the cells: At the end of the final test microscopic observations were performed on the highest test concentration to observe for any abnormal appearance of the algae.
-Recording of Cell Densities: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions. Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.

TEST CONCENTRATIONS
- Solution containing 1.0, 10 and 100 % of the SS prepared at a loading rate of 100 mg/L.
Reference substance (positive control):
yes
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
13 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
(inhibition)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
13 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield inhibition
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 13 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
(inhibition)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 13 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield inhibition
Details on results:
MEASURED CONCENTRATIONS
- Samples taken from the undiluted SS were analysed. The results were, however, not acceptable since they were above the validated range. Consequently, the reserve samples were analysed. The actual exposure concentration was 13 mg/L at the start of the test. This concentration remained stable throughout the test duration, i.e. was 101 % of initial during the test.
- Based on this results, effects parameters were based on the initially measured concentration.


INHIBITION OF GROWTH RATE AND YIELD
- No biologically significant differences were recorded between the values for growth rate at any of the test concentrations when compared to the control group.
- Growth rates were slightly inhibited with increasing concentrations of the test material, up to 5 % at the limit concentration. Even though inhibition at the limit concentration was statistically significant, it was considered not biologically relevant, i.e. effects were <10 %.
- Inhibition of yield increased with increasing concentration of the test material resulting in 25 % inhibition at the limit concentration, which was statistically significant.
- Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the limit concentration when compared to the control.


EXPERIMENTAL CONDITIONS
- The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 unit).
- During the exposure period the temperature measured in the incubator was maintained between 22 and 23 °C. Temperature remained within the limits prescribed by the study plan (21-24 °C, constant within 2 °C).
Reported statistics and error estimates:
- For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the highest test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Two sample t-test Procedure, α=0.05, one-sided, smaller).
- No EC50-values could be calculated because the test material proved to be non-toxic (EC50 > maximum soluble concentration tested > maximum soluble concentration tested).

Acceptability of the Test

- In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 298).

- The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35 % (i.e. 15 %).

- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 % (i.e. 1.4 %).

Table 1: Summary of results for the total test period

Test material concentration

(% SS)

% Inhibition

Growth rate

Yield

1.0

1.06

6.09

10

2.27

12.25

100 (13 mg/L)

4.96*

24.74*

* - effect was statistically significant

 

Table 2: Growth Rate And Percentage Inhibition At Different Time Intervals

Test material concentration

(% SS)

0-24 h

24-48 h

48-72 h

Mean

% Inhibition

Mean

% Inhibition

Mean

% Inhibition

Control

2.211

0.00

1.801

0.00

1.684

0.00

100 (13 mg/L)

2.144

3.03

1.595

11.41

1.673

0.62

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study with Pseudokirchneriella subcapitata, the test material showed no biologically relevant inhibition of growth rate at any of the concentrations tested. Both the EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h EYC50) were beyond the range tested, i.e. exceeded 13 mg/L, being considered the maximum soluble concentration of test material in test medium at a loading rate of 100 mg/L. The 72h-NOEC for growth rate inhibition was 13 mg/L based on biologically relevance. The 72h-NOEC for yield inhibition was lower than 13 mg/L based on both statistically significance and biologically relevance.
Executive summary:

The objectiveofthe study was to evaluate the test material for its ability to generate toxic effects in Pseudokirchneriella subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10and EC50for both inhibition of growth rate and inhibition of yield.The study was performed in accordance with the standardised guideline OECD 201, under GLP conditions.

A Saturated Solution (SS) of the test material was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium.

A combined limit/range-finding test was performed. Six exponentially growing algal cultures per group were exposed to an untreated control and to solutions containing 1.0, 10 and 100 % of the SS prepared at a loading rate of 100 mg/L. The initial algal cell density was 10^4cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

No biologically significant differences were recorded between the values for growth rate at any of the test concentrations when compared to the control group.

Samples taken from the undiluted SS were analysed. The results were, however, not acceptable since they were above the validated range. Consequently, the reserve samples were analysed. The actual exposure concentration was 13 mg/L at the start of the test. This concentration remained stable throughout the test duration, i.e. was 101 % of initial during the test. Based on this results, effects parameters were based on the initially measured concentration.

Under the conditions of this study with Pseudokirchneriella subcapitata, the test material showed no biologically relevant inhibition of growth rate at any of the concentrations tested. Both the EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h EYC50) were beyond the range tested, i.e. exceeded 13 mg/L, being considered the maximum soluble concentration of test material in test medium at a loading rate of 100 mg/L. The 72h-NOEC for growth rate inhibition was 13 mg/L based on biologically relevance. The 72h-NOEC for yield inhibition was lower than 13 mg/L based on both statistically significance and biologically relevance.

Description of key information

Under the conditions of this study with Pseudokirchneriella subcapitata, the test material showed no biologically relevant inhibition of growth rate at any of the concentrations tested. Both the EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h EYC50) were beyond the range tested, i.e. exceeded 13 mg/L, being considered the maximum soluble concentration of test material in test medium at a loading rate of 100 mg/L. The 72h-NOEC for growth rate inhibition was 13 mg/L based on biologically relevance. The 72h-NOEC for yield inhibition was lower than 13 mg/L based on both statistically significance and biologically relevance.

Key value for chemical safety assessment

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

Additional information

The objective of the study was to evaluate the test material for its ability to generate toxic effects inPseudokirchneriella subcapitataduring an exposure period of 72 hours and, if possible, to determine the NOEC, EC10and EC50for both inhibition of growth rate and inhibition of yield. The study was performed in accordance with the standardised guideline OECD 201, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

A Saturated Solution (SS) of the test material was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium.

A combined limit/range-finding test was performed. Six exponentially growing algal cultures per group were exposed to an untreated control and to solutions containing 1.0, 10 and 100 % of the SS prepared at a loading rate of 100 mg/L. The initial algal cell density was 10^4cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

No biologically significant differences were recorded between the values for growth rate at any of the test concentrations when compared to the control group.

Samples taken from the undiluted SS were analysed. The results were, however, not acceptable since they were above the validated range. Consequently, the reserve samples were analysed. The actual exposure concentration was 13 mg/L at the start of the test. This concentration remained stable throughout the test duration, i.e. was 101 % of initial during the test. Based on this results, effects parameters were based on the initially measured concentration.

Under the conditions of this study with Pseudokirchneriella subcapitata, the test material showed no biologically relevant inhibition of growth rate at any of the concentrations tested. Both the EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h EYC50) were beyond the range tested, i.e. exceeded 13 mg/L, being considered the maximum soluble concentration of test material in test medium at a loading rate of 100 mg/L. The 72h-NOEC for growth rate inhibition was 13 mg/L based on biologically relevance. The 72h-NOEC for yield inhibition was lower than 13 mg/L based on both statistically significance and biologically relevance.

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