Registration Dossier

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the following schedule:
Frequency: at 0, 24 and 72 hours
Volume: 3 mL from the appropriate centre of test vessels
Storage: Samples were stored in a freezer (≤-15°C) until analysis.

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 the WAF prepared at a loading rate of 100 mg/L 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. Additionally, reserve samples of 3.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C).

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
The batch of the substance tested was a clear yellow viscous liquid (UVCB) with a purity of 99.2% which 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. Preparation of test solutions was performed under dimmed light conditions with glassware wrapped in aluminium foil to minimize exposure to light.

Preparation of test solutions started with loading rates individually prepared at 1.0, 10 and 100 mg/L. A 31-minute period of ultrasonic waves followed by an overnight period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. The obtained mixtures were centrifuged at 10000 rpm for 10 minutes. Subsequently, the supernatant was drained from the pelleted undissolved material using a pipette and the obtained Water Accommodated Fractions (WAFs) were used as test concentrations. All test solutions were clear and colorless 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

Test organisms

Test organisms (species):
other: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)
Details on test organisms:
TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) - Unicellular green algae
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In house laboratory culture
- Age of inoculum (at test initiation): Not specified
- Method of cultivation: Three 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.

Study design

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

Test conditions

Hardness:
24 mg/L as CaCO3
Test temperature:
Maintained between 23 and 24°C constant to within 1°C.
pH:
Within the limits of 6 to 9 and not varying by more than 1.5 units (test 7.9 to 8.1 and control 8.2 to 8.3)
Dissolved oxygen:
Not specified
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
Nominal: Control, 1, 10 and 100 mg/L
Measured: 0, 0.74, 8.2 and 48 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass vessels with aluminium caps
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 mL vessels filled with 50 mL test or control solutions
- Aeration: No
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 179 x 10^4 cells/mL (mean from all vessels)
- No. of organisms per vessel: Not applicable
- No. of vessels per concentration (replicates): 3 replicates at nominally 1 and 10 mg/L and 6 at nominally 100 mg/L
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes as per OECD guideline (M2 media)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 95 to 97 µE/m2/sec when measured in the photosynthetically effective wavelength of 400 to 700 nm.
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- 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 (path length = 10 mm). Test 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.

- Other: Abnormal appearance of the algae compared to the control was assessed using a microscope at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Combined limit test and range finder
- Justification for using less concentrations than requested by guideline: Combined limit test and range finder
- Range finding study
- Test concentrations: Combined limit test and range finder
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks:
Based on biological relevance
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 48 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks:
Based on biological relevance
Details on results:
Measured test item concentrations:
Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 0.74, 8.2 and 48 mg/L at WAFs prepared at 1.0, 10 and 100 mg/L, respectively. The measured concentrations remained stable throughout the exposure period (102 – 113% relative to initial), except for the WAF prepared at 1.0 mg/L which was measured to be at 122% of initial at the end of the test.

Since no biologically relevant effects (i.e.>10%) were observed at the WAF prepared at 1.0 mg/L of which the actual exposure concentration only slightly exceeded the upper limit of the OECD stability criteria (i.e. 80 -120% of initial), effect parameters were expressed in terms of the initially measured concentrations.

The concentrations measured in the samples taken from solutions with algae were not comparable with the concentrations measured in the samples without algae. However, it can be stated that the presence of the algae had no negative effect on the concentration of the test item in test medium throughout the test. This measured concentration was determined to be the maximum solubility of the potentially soluble constituents of the UVCB substance. As such the EC50 values exceeded the limit of solubility.

Inhibition of growth rate:
Statistically significant growth rate of 9.9% was recorded at the limit concentration after 72 hours of exposure. No biologically relevant growth rate inhibition (i.e. >10%) was measured at any test concentration. 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.
Results with reference substance (positive control):
- Results with reference substance valid?
- EC50:
Algae were exposed for a period of 72 hours to K2Cr2O7 (Potassium dichromate) concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and to a control. The initial cell density was 1.0 x 10^4 cells/mL. The EC50 for growth rate inhibition (72h-ErC50) was 0.90 mg/L with a 95% confidence interval ranging from 0.88 to 0.93 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ErC50 for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
With regards to growth rate, Shapiro-Wilk's test on normal distribution was initially employed followed by Levene's test on variance homogeneity (with residuals). The NOEC was determined by using a two sample t-test procedure.

Any other information on results incl. tables

Growth rate and percentage inhibition for the total test period:

Test item1
Initially measured conc. (mg/L)
 

Mean

Std. Dev.

n

%Inhibition

Control

1.729

0.0136

6

 

0.74

1.714

0.0077

3

0.87

8.2

1.663

0.0432

3

3.8

48

1.558

0.0149

6

 9.9#

1 Test Substance; # effect was statistically significant however biologically not relevant (<10%).

Analytical determinations:

Time of sampling
[hours]

Date of sampling

Date of
analysis1

Loading rate2

[mg/L]

Analyzed concentration
[mg/L]

Relative to
initial
[%]

0

19 Jun 2018

27 Jun 2018

0

n.d.

 

 

 

 

1.0

0.740

 

 

 

 

10

8.21

 

 

 

 

100

48.2

 

 

 

 

 1003

49.9

 

24

20 Jun 2018

27 Jun 2018

0

n.d.

n.a.

 

 

 

1.0

0.756

102

 

 

 

10

8.36

102

 

 

 

100

51.1

106

 

 

 

 1003,4

60.3

121

72

22 Jun 2018

27 Jun 2018

0

n.d.

n.a.

 

 

 

1.0

0.900

122

 

 

 

10

8.34

102

 

 

 

100

54.4

113

 

 

 

 1003

44.0

88

1. Samples were stored in the freezer (≤ -15°C) until the day of analysis.

2. Awater accommodated fraction (WAF)prepared at the loading rate.

3. Without algae.
4. The sample was re-diluted as initially applied dilution factor resulted in concentration above the calibration range. Until analysis, 28 Jun 2018, the sample was kept in the auto sampler at room temperature.

n.d. Not detected.

n.a. Not applicable.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present study with Raphidocelis subcapitata, no biologically relevant inhibition of growth rate was recorded at any of the concentrations of the test substance tested.