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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 April 2016 - 18 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23.
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Remarks:
3 November 2015

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- physical appearance: brown coloured, brittle solid
- storage conditions: at room temperature
Specific details on test material used for the study:
- Solubility in water: 4.21 g/L at 20°C. This should be considered a maximum value, as it might have been biased by the presence of undissolved material (see section 4.8).

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0, t=24 and t=72
Volume: 1.0 mL
Storage: Samples were stored in a freezer until analysis.
In addition, the filter used to prepare the WAF at 10 mg/L was retained for possible analysis of the residue.

- At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: The test item was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/ composition of the test item. All test concentrations were prepared separately and started with loading rates ranging from 1.0 to 100 mg/L for the range-finding test and from 0.10 to 10 mg/L for the final test. Three days of magnetic stirring was applied to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixtures were filtered through a 0.45 μm membrane filter where after the clear and colourless Water Accommodated Fractions (WAFs) were used for testing.
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.
- Controls: test medium without test item or other additives (blank-control)

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture.

CULTIVATION:
- Method: 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.
- Light intensity: 60 to 120 µE/m^2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Medium different from test medium: yes, M1

- 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.
- Pre-culture medium different from test medium: no, M2

ACCLIMATION
- Acclimation period: no

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
24 mg CaCO3 mg/L
Test temperature:
21 - 23 °C
pH:
7.8-7.9 throughout the test
Nominal and measured concentrations:
Nominal test concentrations: WAFs prepared at 0.10, 0.32, 1.0, 3.2 and 10 mg/L
Measured test concentrations: 0.025, 0.084, 0.084, 0.097 and 0.99 mg/L based on Time Weighted Average for 0.10, 0.32, 1.0, 1.0 (without algae) and 3.2 mg/L. No sample was analysed for the nominal concentration of 10 mg/L. For details on measured concentrations see table 1 and details on results.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, open, fill volume: 50 mL
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 158.6 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Standard test medium used: yes, M2
- Source of dilution water: Milli-RO water
- Culture medium different from test medium: yes, M1

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: continuous illumination using TLD-lamps with a light intensity within the range of 68 to 78 µE/m^2/s

EFFECT PARAMETERS MEASURED: growth rate and yield at 72 hours.
- Additional measurements: pH at the beginning and at the end of the test. temperature of the medium continuously in a temperature control vessel, appearance of the cells at the end of the final test by microscopic observations.
- Determination of cell concentrations: 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 immersion probe (pathlength =20 mm). Algal medium was used as blank. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.

RANGE-FINDING STUDY
- Test concentrations: WAFs prepared at 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes, a 54.7% inhibition of growth rate at 1.0 mg/L resulted in an expected EL50 for growth rate inhibition of approximately 1 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (July 2016)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
1.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- No biological, behavioural or other abnormalities were observed.

Analysis of the samples taken at the start of the test showed measured concentrations of 0.033, 0.11, 0.15 and 1.1 mg/L in WAFs at 0.10, 0.32, 1.0 and 3.2 mg/L, respectively, based on one component of the test item. Only the 1.1 mg/L concentration remained stable during the test period (96% of initial after 24 hours of exposure and 89% of initial at the end of the test). The 0.033 and 0.11 mg/L concentrations remained stable during the first 24 hours of exposure (82-87% of initial) but decreased to 59-60% at the end of the test. The 0.15 mg/L concentration obtained in the WAF prepared at 1.0 mg/L with algae decreased more and more rapidly than the other two lower concentrations, i.e. to 60% of initial after 24 hours of exposure and further to 33% of initial at the end of the test. The measured concentration in the samples taken from the solution without algae (WAF at 1.0 mg/L) was similar to the concentration in the WAF at 1.0 mg/L with algae at the start of the test but remained stable during the first 24 hours of exposure (84% of initial) and decreased to 70% of initial at the end of the test. These concentrations were therefore more in line with the analysis of the other WAFs with algae than with the concentrations measured in the WAF at 1.0 mg/L with algae. The concentrations measured in the WAF at 1.0 mg/L with algae were therefore considered as outliers although a reason for the deviating measured concentrations could not be found. When calculating the Time Weighted Average (TWA) exposure concentrations exposure concentrations seemed to be the same in the WAFs at 0.32 and 1.0 mg/L. This however was not in line with the effects observed on algal growth. Therefore, the effect parameters were based on the loading rates rather than the measured concentrations.

Individual pH and temperature values remained within acceptable limits throughout the duration of the study.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50: 1.0 mg/L (95%-cI: 0.97-1.0 mg/L
- Other: results fell within the historical range.
Reported statistics and error estimates:
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

Table 1 Measured concentrations versus nominal concentrations

Test item

WAF at x mg/L

Measured concentration (mg/L)

TWA (mg/L)

 

t=0h

t=24h

t=72h

0.10

0.0325

0.0267

0.019

0.025

0.32

0.106

0.0921

0.0635

0.084

1.0

0.152

0.0915

0.0499

0.084

1.0*

0.119

0.0996

0.0837

0.097

3.2

1.05

1.01

0.933

0.99

10

-

-

-

-

* Without algae

- Not measured

Table 2 Percentage inhibition of growth rate (total test period) during the final test

Treatment

WAF at x mg/L

Mean

Std. Dev.

N

% inhibition

Control

1.686

0.0425

6

 

0.10

1.652

0.0347

3

2.0

0.32

1.600

0.0166

3

5.1*

1.0

1.132

0.0279

3

32.9*

3.2

0.000

0.0000

3

100.0*

10

0.182

0.1190

3

89.2*

* Effect was statistically significant

Table 3 Effect parameters

 

Parameter (mg/L)

NOELR

EL10

EL20

EL50

Growth rate

Value

0.10

0.73

0.86

1.2

Lower 95%-cI

0.21

0.47

1.1

Upper 95%-cI

0.84

0.92

2.3

Yield

Value

0.10

 

0.20

0.28

0.52

Lower 95%-cI

0.18

0.26

0.49

Upper 95%-cI

0.22

0.30

0.56

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For details on validity criteria see 'overall remarks' section.
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, the test material inhibited growth rate of freshwater algae significantly at loading rates of 0.32 mg/L and higher. The EL50 for growth rate inhibition (72h-EL50) was 1.2 mg/L with a 95% confidence interval ranging from 1.1 to 2.3 mg/L. The EL50 for yield inhibition (72h-EL50) was 0.52 mg/L with a 95% confidence interval ranging from 0.49 to 0.56 mg/L. The 72h-NOEC for inhibition of growth rate was 0.10 mg/L.