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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:
2017-07-17 to 2017-08-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: 10 mL of the test item concentrations and the control were filled into a vial.
- Sample storage conditions before analysis: All samples were stored at 6 +- 2 °C until sample preparation and at room temperature until the start of the analysis, if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal loading of 4.37 mg/L test item was prepared with dilution water one day prior to the start of exposure. This dispersion was stirred for 24 hours with 1100 rpm at room temperature. After a separation phase of 1 hour, the saturated solution was removed by siphoning (from the approximate bottom of the flask) and used in the test.
- Controls: Negative control with test water but witout test item; positive control with the reference substance potassium dichromate
- Evidence of undissolved material: The solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: SAG 61.81
- Source: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum: 4 days old preculture, prepared in dilution water
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2590 - 5180 lux for 24 hours per day
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol Ca+Mg/L (nominal, test medium)
Test temperature:
22.5 °C (mean)
pH:
8.06 to 9.42
Nominal and measured concentrations:
Nominal loading [mg/L]: 4.37; 0 (control), 2.56, 6.40, 16.0, 40.0, and 100% of the saturated solution
Geometric mean measured test item concentrations [µg/L]: 491, 125, 53.0, 21.7, 7.29, 0 (control)
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile headspace flasks
- Type: closed
- Volume, seals: Nominal volume = 59 mL; fill volume = 59 mL; with aluminium tops with PTFE seals
- Aeration: No, but agitation on a rotary shaker and oscillated at approx. 70 rpm
- Initial cells density:
Nominal: Approx. 5 x 10^3 - 10^4 cells/mL
Actual: 6839 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: Yes (OECD TG 201 medium), but additionally compounds were added to enable sufficient growth under conditions without headspace (NaHCO3, MES monohydrate)

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approx. 4440 to 8880 lux, within +- 15% over incubation area

EFFECT PARAMETERS MEASURED:
- Chlorophyll measurement: Chlorophyll a -fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal.
- Other: Microscopic evaluation for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: Yes
- Test concentrations: 3.10, 0.335 and 0.0309 mg/L (at the start of the range finding test)
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
465 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits: 133 - 491 µg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
264 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits: 51.9 - 491 µg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
147 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
biomass
Remarks on result:
other: 95% confidence limits: 80.2 - 334 µg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
13 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
biomass
Remarks on result:
other: 95% confidence limits: < 7.29 - 47.2 µg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
7.29 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: growth rate and biomass
Details on results:
- Exponential growth in the control: Yes
- Observation of abnormalities: No
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: Yes, at the 7.29 µg/L level.
- Effect concentrations exceeding solubility of substance in test medium: Yes, in one replicate at the 7.29 µg/L level (geometric mean measured concentration).
Results with reference substance (positive control):
48h ErC50 (growth rate) = 0.498 mg/L;
48h EyC50 (yield) = 0.320 mg/L
Reported statistics and error estimates:
ECx-values with confidence intervals of grwoth rate and ield inhibition after 72 hours were calculated by sigmoidal dose-response. The NOEC and LOEC were determined by calculation of statistically significant differences of grwoth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT's test were used for NOEC/LOEC calculations.

Table 1: Measured concentrations and cercentage of the initial concentration of the test item.

Sampling date 2017-08-15
0 h
2017-08-18
72 h
Geometric mean
Start of analysis 15.08.2017 18.08.2017
Dilution level of the saturated solution [%]  TEST ITEM
Meas. conc. [µg/L] Meas. conc. [µg/L] [%] [µg/L]
100 571 421 74 491
40 159 97.6 61 125
16 66.2 42.4 64 53
6.4 26.8 17.6 66 21.7
2.56 10.6 6.701) 63 7.29
Control < LOQ < LOQ < LOQ

Meas. conc. = Measured concentration of the test item

% = Percentage of the nominal concentration (10 µg test item/L)

LOQ = Measured concentration < 70% LOQ, therefore 1/2 LOQ (5 µg test item /L) taken into account for geometric mean

Table 2: Evaluation after 72 hours. Statistically significant differences of growth rate and yield compared to control values are marked (+), not significant diffrerences are marked (-).

Geometric mean measured test item concentration [µg/L] Replicate No. Growth rate [d-1] Inhibition of growth rate [%] Yiels [cells/mL] Inhibition of yield [%]
491 1 0.659 58 42582 94
2 0.662 58 43056 94
3 0.703 55 49528 93
Mean (+) 0.675 57 (+) 45055 94
125 1 1.46 7 535382 27
2 1.46 7 540707 27
3 1.46 5 594107 19
Mean (+) 1.47 6 (+) 556732 25
53 1 1.44 8 503752 32
2 1.47 6 549076 26
3 1.46 7 534826 28
Mean (+) 1.45 7 (+) 529218 28
21.7 1 1.39 11 433528 41
2 1.43 9 487893 34
3 1.42 9 473468 36
Mean (+) 1.41 10 (+) 464963 37
7.29 1 1.52 2 655465 11
2 1.56 1 718403 3
3 1.57 0 745703 -1
Mean (-) 1.55 1 (-) 706524 4
Control 1 1.56 737042
2 1.61 843256
3 1.55 699443
4 1.55 717467
5 1.56 734204
6 1.54 695200
Mean 1.56 737769

negative values = growth stimulation

Validity criteria fulfilled:
yes
Conclusions:
The 72h ErC10 and the ErC50 (growth rate) of the test item was determined to be 0.264 and 0.465 mg/L, respectively. The 72h EyC10 and the EyC50 (yield) of the test item was determined to be 0.013 and 0.147 mg/L, respectively.
Executive summary:

To assess the ecotoxicological potential of the test item to aquatic algae (Pseudokircheriella subcapitata), an algal inhibition test according to OECD Guideline 201 and EU Method C.1 was carried out in compliance with GLP principles. The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A saturated solution with a nominal loading of 4.37 mg test item /L was freshly prepared with dilution water one day prior to the start of exposure. This saturated solution was stirred for 24 hours with 1100 rpm at room temperature. The saturated solution and four dilution levels out of the stock solution were tested in geometrical with a dilution factor of 2.5. The geometric mean measured concentrations (analyzed by GC-MS) were 491, 125, 53.0, 21.7 and 7.29 µg/L. Three replicates were tested for each test item concentrations and six replicates for a negative control (with test media and inoculum, but without the test item). The suitability of the test system was confirmed by a positive control with the reference substance potassium dichromate. The effect values given are based on geometric mean measured concentration. In result, the 72h ErC10 and the ErC50 (growth rate) of the test item was determined to be 0.264 and 0.465 mg/L, respectively. The 72h EyC10 and the EyC50 (yield) of the test item was determined to be 0.013 and 0.147 mg/L, respectively. The 72h NOEC values for both growth rate and yield after 72 hours were 7.29 µg/L (0.00729 mg/L). For the reference substance, the 48h ErC50 for growth rate and yield was determined to be 0.498 mg/L and 0.320 mg/L, respectively. All validity criteria of the guidelines were fulfilled.

Description of key information

The 72h ErC10 and the ErC50 (growth rate) of the test item was determined to be 0.264 and 0.465 mg/L (geometric mean measured concentration), respectively. The 72h EyC10 and the EyC50 (yield) of the test item was determined to be 0.013 and 0.147 mg/L (geometric mean measured concentration), respectively.  

Key value for chemical safety assessment

EC50 for freshwater algae:
0.465 mg/L
EC10 or NOEC for freshwater algae:
0.264 mg/L

Additional information

To assess the ecotoxicological potential of the test item to aquatic algae (Pseudokirchneriella subcapitata), a valid Algal Inhibition Test according to OECD Guideline 201 and EU Method C.1 was carried out in compliance with GLP principles. The study was conducted under static conditions with an initial cell density of 6839 cells/mL. The geometric mean measured concentrations (analyzed by GC-MS) were 491, 125, 53.0, 21.7and 7.29 µg/L. Three replicates were tested for each test item concentration and six replicates for a negative control (with test media and inoculum, but without the test item). In result, the 72h ErC10 and the ErC50 (growth rate) of the test item was determined to be 0.264 and 0.465 mg/L, respectively. The 72h EyC10 and the EyC50 (yield) of the test item was determined to be 0.013 and 0.147 mg/L, respectively. The 72h NOEC values for both growth rate and yield after 72 hours were 7.29 µg/L (0.00729 mg/L).

The effect values are based on geometric mean measured concentrations.