Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 January 2018 to ****
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)
Specific details on test material used for the study:
Purity: >99%
Description: Clear amber viscous liquid
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Samples (5 mL) of test media of freshly prepared test media (0 hour) and pooled expired test media (72 hours) were collected in duplicate.
- Sample storage conditions before analysis: Storage stability was not assessed. Samples were extracted and analysed on the day of receipt or sample extracts were stored refrigerated.
- Sample processing: Concentrations of the test substance in OECD medium were determined based on dipentylamine (formed in situ from the acid-catalysed degradation of the test substance) as a marker for quantification. Aqueous samples were treated with tin (II) chloride in hydrochloric acid (following dilution with OECD medium if required to bring the response within the calibration range), then treated with acetonitrile.
Vehicle:
no
Details on test solutions:
The dilution media used was OECD medium.

The test substance is a complex multi-constituent substance with low water solubility and as such falls into the category of a “difficult substance” as defined by the OECD Guidance Document (OECD Series on Testing and Assessment, No. 23 (2000); Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures). Based on the recommendations of this guidance, the test solutions were prepared as Water Accommodated Fractions (WAFs).

At the start of the test, ca 10, 18, 32, 56 and 100 mg of test substance were each separately weighed onto glass slides. The slides were separately added to 1000 mL of OECD medium and stirred for ca 23 hours at a rate such that a slight dimple was formed at the surface. After the stirring period, the preparations were allowed to settle for ca 1 hour after which the aqueous phase was removed by careful mid-depth siphoning to give the 10, 18, 32, 56 and 100 mg/L loading rate WAFs. A control treatment was prepared using the same preparation method without the addition of test substance.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Test type:
static
Water media type:
other: OECD medium
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not specified
Test temperature:
21.4 – 23.7 °C
pH:
6.73 - 9.31
Dissolved oxygen:
Not specified
Salinity:
Not applicable
Conductivity:
Not specified
Nominal and measured concentrations:
The definitive test was conducted at nominal loading rates of 10, 18, 32, 56 and 100 mg/L, with a concurrent control group.

Analysis of the freshly prepared samples at 0 hours resulted in measured concentrations 0.0500, 0.0909, 0.0965, 0.167 and 0.241 mg/L for the 10, 18, 32, 56 and 100 mg/L loading rate WAFs, respectively. The corresponding pooled old media samples at 72 hours resulted in measured concentrations of 0.0473, 0.0812, 0.0909, 0.146 and 0.170 mg/L, respectively.

Given that the test substance is a multi-constituent substance, and analytical monitoring was limited to the dipentylamine fraction of the test substance, the effect concentrations are based on nominal loading rates.
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile, autoclaved glass 250 mL Erlenmeyer (conical) flasks
- Fill volume: 100 mL
- Aeration: None; shaken continuously (100 rpm) to keep algae in suspension and facilitate transfer
of CO2.
- Renewal rate of test solution: Not applicable; static
- Initial cell density: 1 x 10^4 cells/mL
- Control end cell density: 97.5 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: Yes; OECD medium

OTHER TEST CONDITIONS
- Intervals of water quality measurement: 0 and 72 hours; temperature also was recorded continuously using minimum/maximum thermometer.
- Sterile test conditions: Yes
- Adjustment of pH: None reported.
- Photoperiod: Constant light
- Light intensity and quality: 6620 – 8530 lux (0 hour); 7360 – 8730 lux (72 hours); under florescent lights, emitting white light across the visible portion of the spectrum (400 - 700 nm).

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: particle counter (Z2 Coulter Counter)
- Chlorophyll measurement: No
- Cell condition: At the end of the test, a subsample of the pooled replicates from the control and each test concentration was taken and the shape and general condition of the algae in each test group was assessed by microscopic observations.

RANGE-FINDING TEST
A range-finding test was conducted at nominal test substance loading rates of 1.0, 10 and 100 mg/L. A concurrent control group was also included. Duplicate test vessels were prepared for the control and each loading rate.
Reference substance (positive control):
yes
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
/ loading rate WAF
Basis for effect:
other: yield
Remarks on result:
other: 95% confidence limits (26 mg/L – not calculable)
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
/ loading rate WAF
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
/ loading rate WAF
Basis for effect:
other: yield and growth rate
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
34 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
/ loading rate WAF
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits (6.3 – 54 mg/L)
Details on results:
- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): None reported
- Test media descriptions: The freshly prepared test media at 0 hours appeared as colourless solutions. The appearance of the test media following the 72-hour exposure period was not observed in error. This deviation to the protocol was not considered to have had an adverse impact on the integrity of the test.
- Any stimulation of growth found in any treatment: Yes
- Effect concentrations exceeding solubility of substance in test medium: Yes
Results with reference substance (positive control):
A positive control study was conducted to determine the effects of the reference substance, potassium dichromate, on algal growth by exposing the green alga, Pseudokirchneriella subcapitata, at the exponential growth phase to the reference substance for 72 hours. The 72-hour EC50 values for yield, area under the growth curve and growth rate were determined to be 0.625, 0.706 and 1.16 mg/L, respectively. The 72-hour NOEC value for yield, area under the growth curve and growth rate was determined to be 0.32 mg/L.

These results were within the expected toxicity range (growth rate EC50: 0.38 - 2.60 mg/L) for this reference substance and all validity criteria were met; the test is therefore considered valid.
Reported statistics and error estimates:
Statistical analysis was performed using the CETIS program v 1.8.6.8.

The average specific growth rate (µ) and the final yield over specified time intervals were analysed using Dunnett Multiple Comparison Test to determine the no observed effect loading rate (NOEL).

Linear interpolation analysis was performed in order to estimate EL10, EL20 and EL50 values. Where possible, 95% confidence limits were calculated for the EL10, EL20 and EL50 values.

Percent inhibition of growth rate ofPseudokirchneriella subcapitata

Nominal Loading Rate

Average specific growth rate, “µ”
(cells x 10^-2/mL)

Inhibition of average specific growth
 rates relative to the control (%)

(mg/L)

0-24
hours

24-48
hours

48-72
hours

0-72
hours

0-24
hours

24-48
hours

48-72
hours

0-72
hours

Control

5.73

6.78

6.53

6.35

NA

NA

NA

NA

10

5.54

7.07

6.90

6.50

3

-4

-6

-2

18

5.19

6.23

6.72

6.05

9

8

-3

5

32

4.87

5.91

6.82

5.87

15

13

-4

8

56

3.81

4.55

5.89

4.75

34

33

10

25

100

4.31

5.40

5.56

5.09

25

20

15

20

 NA : Not applicable

Negative percentage inhibition values relative to the control indicate growth stimulation and are not considered an adverse effect.

 

 

Percent inhibition of yield of Pseudokirchneriella subcapitata

Nominal Loading Rate
(mg/L)

Initial
cell density
(cells x 10^4/mL)

Yield at
72 hours *
(cells/mL)

Mean
Final Yield
per treatment
(cells x 10^4/mL)

% Inhibition
in Final Yield
relative to
control

Control

1

1198420
765700
1043620
905340
864000
1012880

96.5

NA

10

1

1049160
1153080
1015480

107

-11

18

1

956220
759480
619640

77.8

19

32

1

492060
743520
833060

69.0

29

56

1

523700
239440
202360

32.2

67

100

1

129760
500420
821320

48.4

50

 *      Final yield (cells/mL) = cell density (cells/mL) at 72 hours – initial cell density (cells/mL)

NA : Not applicable

Negative percentage inhibition values relative to the control indicate growth stimulation and are not considered an adverse effect.

Validity criteria fulfilled:
yes
Remarks:
The control cell densities increased by a factor of 96. The mean coefficient of variation (CV) for section-by-section specific growth rate in the control cultures was 12% and the overall CV for specific growth rate was 3.42%.
Conclusions:
Based on nominal loading rates, the 72-hour EyL50 and the 0-72 hour ErL50 values were calculated to be 46 and >100 mg/L loading rate WAF, respectively. The corresponding No Observed Effect Loading rate (NOEL) for yield and specific growth rate after 72 hours was 32 mg/L loading rate WAF.
Executive summary:

The study was conducted to determine the effects of the test substance on the growth of the freshwater green alga, Pseudokirchneriella subcapitata, during a 72-hour growth inhibition toxicity test. The test was conducted in accordance with OECD Chemicals Testing Guideline No. 201. Freshwater Alga and Cyanobacteria, Growth Inhibition Test (adopted 23 March 2006, Annex 5 corrected 28 July 2011).

The test substance is a complex multi-constituent substance with low water solubility and as such falls into the category of a “difficult substance” as defined by the OECD Guidance Document (OECD Series on Testing and Assessment, No. 23 (2000); Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures). Based on the recommendations of this guidance, the test solutions were prepared as Water Accommodated Fractions (WAFs).

The definitive test was conducted at nominal loading rates of 10, 18, 32, 56 and 100 mg/L.The range was based on the results of a range‑finding test, for which only the key results have been reported.A concurrent control group was also included.

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 x 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.

Samples of test media were analysed at 0 (new media) and 72 hours (expired test media). Concentrations of the test substance were determined by liquid chromatography triple quadrupole mass spectrometry (LC-TQMS) systembased on quantitation ofthe marker analyte,dipentylamine (formed in situ from the acid-catalysed degradation of the test substance).

Analysis of the freshly prepared samples at 0 hours resulted in measured concentrations 0.0500, 0.0909, 0.0965, 0.167 and 0.241 mg/L for the 10, 18, 32, 56 and 100 mg/L loading rate WAFs, respectively. The corresponding pooled old media samples at 72 hours resulted in measured concentrations of 0.0473, 0.0812, 0.0909, 0.146 and 0.170 mg/L, respectively. Given that the test substance is a multi-constituent substance, and analytical monitoring was limited to the dipentylamine fraction of the test substance, the effect concentrations are based on nominal loading rates.

Based on nominal loading rates, the 72-hour EyL50 and the 0-72 hour ErL50 values were calculated to be 46 and >100 mg/L loading rate WAF, respectively. The corresponding No Observed Effect Loading rate (NOEL) for yield and specific growth rate after 72 hours was 32 mg/L loading rate WAF.

Description of key information

In an OECD 201 study, conducted according to GLP, the effect of the test substance on the growth of the freshwater green algae,Pseudokirchneriella subcapitata,was investigated and the 72-hour EL50 value based on growth rate was determined to be greater than 100 mg/L loading rate WAF and the No Observed Effect Loading rate (NOEL) was determined to be 32 mg/L loading rate WAF (Smithers Viscient (ESG) Ltd., 2018).

Key value for chemical safety assessment

Additional information