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Ecotoxicological information

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:
21-03-2016 to 21-10-2016
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)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
All concentration levels and the control were analytically verified via LC-MS/MS after 0 hours (start of the exposure) and 72 hours (end of the exposure).
For the test item analysis at the beginning of the exposure, separate replicates for each test item concentration and control were prepared with algae. For the test item analysis after 72 hours, samples were taken from the test replicates.
Vehicle:
no
Details on test solutions:
The test item is known to degrade quickly (half-life < 1 hour) by hydrolysis. Therefore, the hydrolysis product was tested as recommended in OECD Guidance Document No. 23 (2000). For this purpose, a stock solution of 100 mg/L of the test item was prepared with dilution water and stirred for 24 hours with 1100 rpm at room temperature before the start of the exposure phase to ensure complete hydrolysis of the test item. Thereafter, undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). During filtration the filter was always kept covered. The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL filtrate were discarded. The filtration was interrupted for ca. 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate (visually clear and colourless) was used in the test. Further dilution levels were prepared from the saturated solution
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata is a suitable green alga species according to the guideline.

Origin Sammlung von Algenkulturen (SAG)
Pflanzenphysiologisches Institut der Universität Göttingen
Nikolausberger Weg 18, D-37073 Göttingen

Cultivation at test facility: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE·m-2·s-1 for 24 hours per day.

A four days old preculture, prepared in dilution water, was used as inoculum.
Initial cell density Nominal: approximately 5 x 103 - 104 cells/mL
Current: 6305 cells/mL
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
Nominal range: 21 - 24 °C, controlled at ± 2 °C
Room temperature [°C]: min.: 22.5 max.: 23.5 mean value: 23.0
pH:
The pH-value at the start of exposure was measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-value was measured from pooled samples of each test item concentration and the control.

Geometric mean measured
test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
19.2 7 47 8.06
7.92 7.52 8.28
3.19 7.63 8.46
1.21 7.72 8.60
0.503 7.87 8.89
Control 7.94 9.05
Nominal and measured concentrations:
The measured concentrations of the test item at the start of the exposure reflect the dilution factor of 2.5. At the end of the exposure, the measured concentrations of the test item were within the range of 86 to 95% of the initially measured concentrations.
5 concentrations were tested in a geometrical series with a dilution factor of 2.5: 2.56 - 6.40 - 16.0 - 40.0 - 100 % of the saturated solution. These dilution levels are corresponding to geometric mean measured test item concentrations of 0.503 - 1.21 - 3.19 - 7.92 - 19.2 mg/L. The concentrations were based on the results of a preliminary range finding test (non-GLP).


Sampling date 2016-04-19 2016-04-22
0 hours 72 hours
Start of the exposure End of the exposure
Dilution level of
the saturated solution Meas.conc. Meas.conc. Geometric mean measured concentrations
[%] [mg/L] [mg/L] [%] [mg/l]

100* 20.1 18.3 91 19.2
40 8.55 7.33 86 7.92
16.0 3.27 3.11 95 3.19
6.40 1.28 1.15 90 1.21
2.56 0.523 0.484 93 0.503
Control
Details on test conditions:
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the definitive test at the saturated solution.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 19.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.19 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
NOEC, LOEC, ECx-values and 95 % Confidence Intervals of the test item (0 - 72 hours) based on geometric mean measured test item concentrations [mg/L]

Growth Rate Inhibition
Geometric mean measured test item concentration [mg/L]
NOEC 3.19
LOEC 7.92
ErC10 10.1 (3.61 – 18.9)
ErC20 13.1 (8.42 – 19.0)
ErC50 > 19.2

Yield Inhibition
Geometric mean measured concentration [mg/L]
NOEC 3.19
LOEC 7.92
EyC10 4.70 (2.12 – 6.97)
EyC20 7.05 (4.10 – 13.3)
EyC50 12.4 (9.41 – 16.8)
Results with reference substance (positive control):
EC50-Values of the Reference Item based on nominal concentrations (mg/L), (0-72 hours)

Growth Rate inhibition Current Study Valid Range (average ± 3 x SD)
ErC50 0.564 0.797 ± 0.428
95 % confidence interval 0.546 - 0.582

Results of the Range Finding Test (0 - 72 hours)

Saturated solution

[%]

Growth Rate Inhibition
[%]

Yield Inhibition
[%]

100

100

100

10

7

33

1

0

-2

Validity criteria fulfilled:
yes
Conclusions:
In this study, teh test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on geometric mean measured test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 19.2 and 12.4 (9.41 – 16.8) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 3.19 mg/L.
Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009/Method C.3.

The study was conducted under static conditions with an initial cell density of 6305 cells/mL. A saturated solution of 100 mg/L was prepared and five dilution levels were tested in a geometrical series with a dilution factor of 2.5:2.56 - 6.40 - 16.0 - 40.0 - 100% of the saturated solution. These dilution levels are corresponding to geometric mean measured test item concentrations of 0.503 - 1.21 - 3.19 - 7.92 - 19.2 mg/L.

Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.The test media were clear throughout the test period.

 

The concentrations of the test item in all test concentrations and the control were analytically verified by LC-MS/MS at the start (0 hours) and at the end (72 hours) of the exposure.The measured concentrations in the fresh media (0 h) were in the range of 0.523 to 20.1 mg test item/L. The measured concentrations in the old media (72 h) were in the range of 0.482 to 18.3 mg test item/L.

 

All effect values are based on the geometric mean measured test item concentrations of the test item.

Description of key information

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009/Method C.3.

The study was conducted under static conditions with an initial cell density of 6305 cells/mL. A saturated solution of 100 mg/L was prepared and five dilution levels were tested in a geometrical series with a dilution factor of 2.5: 2.56 - 6.40 - 16.0 - 40.0 - 100% of the saturated solution. These dilution levels are corresponding to geometric mean measured test item concentrations of 0.503 - 1.21 - 3.19 - 7.92 - 19.2 mg/L.

Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditionswere within the acceptable limits.The test media were clear throughout the test period.

 

The concentrations of thetest itemin all test concentrations and the control were analytically verified by LC-MS/MS at the start (0 hours) and at the end (72 hours) of the exposure.The measured concentrations in the fresh media (0 h) were in the range of 0.523 to 20.1 mg test item/L. The measured concentrations in the old media (72 h) were in the range of 0.482 to 18.3 mg test item/L.

 

All effect values are based on the geometric mean measured test item concentrations of the test item.

Key value for chemical safety assessment

EC50 for freshwater algae:
19.2 mg/L
EC10 or NOEC for freshwater algae:
3.19 mg/L

Additional information