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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:
2016-07-18 to 2016-07-21
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)
Version / remarks:
2011
Deviations:
yes
Remarks:
natural river water was used to represent a more natural scenario; detailed analysis were provided and results were considered not to be reliable.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Test item: PC-2015-612
Batch number: E815300321
CAS number: 554-62-1
Chemical name: 1,3,4-Octadecanetriol, 2-amino- (2S,3S,4R)-
Expiry date: 2018-03-10
Analytical monitoring:
yes
Details on sampling:
Sampling
All concentration levels and the controls 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 the controls were prepared with algae. For the test item analysis after 72 hours, samples were taken from the test replicates.
Vehicle:
yes
Remarks:
Dimethylformamide (DMF)
Details on test solutions:
Stock solutions/ Preparation of the Test Concentrations
For the preparation of the test concentrations of the test item, appropriate stock solutions were prepared in DMF (solvent). The stock solutions were prepared in measuring flasks (10 mL capacity) by direct weighing of appropriate amounts of the test item into the measuring glass flasks. Thereafter, the flasks were filled up with DMF to 10 mL. The stock solutions were treated with ultrasound for 15 minutes at room temperature. The measuring glass flasks containing the stock solutions prepared with DMF were closed and opened only for taking aliquots for preparation of the test concentrations. The test concentrations were prepared by adding 0.05 mL/L of the appropriate stock solutions to the natural river water. These solutions were stirred for 30 minutes at 1100 rpm at room temperature and used for testing.

Test concentrations (nominal)
Five concentration levels were tested in a geometrical series with a dilution factor of 2: 62.5 — 125 — 250 — 500 — 1000 pg/L. The concentration levels are based on the results of a preliminary range finding test (non-GLP).

Control
Six replicates (without test item) were exposed under the same conditions as the test concentrations.

Solvent control
Additionally, a solvent control with 0.05 mL DMF/L in test medium was prepared and tested under the same conditions as the test group. With regard to the low water solubility of the test item DMF was used as a solvent.

Supplier: Merck
Purity: 99.8 %.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algea
- Strain: Pseudokirchneriella subcapitata HINDAK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universitat Gottingen
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 pE •m-2 .s-1 for 24 hours per day.
- Culture medium: Nutrient medium Z according to LOTTGE et al. (1994)
- Age of inoculum (at test initiation): A three days old preculture, prepared in test medium was used as inoculum.


ACCLIMATION
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Remarks:
natural river water
Limit test:
no
Total exposure duration:
72 h
Hardness:
160 [mg CO3/L]
Test temperature:
22.0 - 22.5 °C
pH:
start: 8.07 - 8.11
end: 7.94 - 8.12
Dissolved oxygen:
8.62 [mg 02/L]
Nominal and measured concentrations:
Nominal: 62.5 — 125 — 250 — 500 — 1000 µg/L
Measured (geometric mean): 8.27 — 12.7 — 33.5 — 63.7 — 176 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks (vol. 250 mL) with cotton wool plugs
- Test volume: 100 mL
- Initial cells density:
Nominal: approximately 5 x 103 - 104 cells/mL
Current: 6305 cells/mL
- Control end cells density: 1531545 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Dilution water: Natural River Water / Supplemental Dilution Water (OECD TG 201 medium)
- Culture medium different from test medium: yes

Water of the river Innerste was used as its properties in terms of organic matter content and suspended solids are considered typical for European rivers and are close to those in the EU risk assessment methods (for details see any other informatin on materials and methods). The river water was supplemented with 50% of the standard OECD medium to enable a sufficient growth of the algae. The supplemental dilution water compounds were applied in a concentrated solution (6.5 mL/L river water) in order to avoid changes of the parameters of the river water.

- Intervals of water quality measurement: The pH-value at the start of exposure was measured in one additional replicate of each test item concentration and the controls. At the end of exposure, the pH-value was measured from pooled samples of each test item concentration and the controls. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Fluorometer: Microplate Reader Chameleon V (HoEx) with Software
- Chlorophyll measurement: 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 each concentration level.
- Microscopic evaluation: Microscopic evaluation of the cells at the start and the end of exposure was carried out. The cells were checked 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
- Range finding study: yes
- Test concentrations: 10 - 100 - 1000 µg/L
- 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:
NOEC
Effect conc.:
12.7 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
33.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
45.5 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95%CI: 38.8 - 51.3
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
166 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95%CI: 152 - 174
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
33.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
50.1 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95%CI: 41.3 - 64.0
Details on results:
During the 72h exposure period, inhibition of growth and inhibition of yield were observed in a dose-response relationship. No adverse effects were observed in the two lowest test concentration, i.e. 8.27 and 12.7 µg/L. The derived 72h-ErC50 was 166 µg/L, 72h-ErC10 was 45.5 µg/L and 72h-NOErC was 12.7 µg/L. The derived 72h-EbC50 was 50.1 µg/L, 72h-EbC10 was18.8 µg/L and 72h-NOEbC was 12.7 µg/L. Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities. All effect values were given based on the geometric mean measured test item concentrations.

Raw data on growth inhibition effect and yield inhibition are presented in any other information on results.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no; the test media were clear throughout the exposure phase.
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
The toxicity of potassium dichromate (SIGMA-ALDRICH, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2016-04-26 to 2016-04-29 (with headspace). The reference item toxicity is in the valid range following test facility SOPs.

ErC50 = 0.460 (95 % confidence interval:0.443 — 0.481)
EbC50 = 0.317 (95 % confidence interval:0.208 — 0.347)
Reported statistics and error estimates:
Statistical Significance (NOEC/LOEC)
One Way Analysis of Variance with Multiple Comparisons versus Control Group (Dunnett's Method); a priori normality check with Normality Test (Shapiro-Wilk) and Equal Variance Test.

Statistical Significance (Control / Solvent Control)
t-test; a priori Normality Test (Shapiro-Wilk) Passed and Equal Variance Test.

Summary table of results after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Geometric mean
measured test item
concentration
[pg/L]

Replicate Growth rate Inhibition of Yield Inhibition of
yiel
d
No. [d-1] [%] [cells/mL] [%]
176 1 0.881 54 82318 96
2 0.901 53 87657 96
3 0.915 53 91694 96
Mean (+) 0.899 53 (+) 87223 96
63.7 1 1.59 18 733989 64
2 1.67 13 933856 54
3 1.65 14 893770 56
Mean (+) 1.64 15 (+) 853872 58
33.5 1 1.78 7 1316450 36
2 1.81 6 1448492 29
3 1.74 9 1172794 43
Mean (+) 1.78 8 (+) 1312579 36
12.7 1 1.95 -1 2154088 -5
2 1.95 -1 2210186 -8
3 1.94 -1 2120768 -4
Mean (-) 1.95 -1 (-) 2161681 -6
8.27 1 2.02 -5 2671149 -31
2 1.94 -1 2112874 -3
3 1.92 0 2009304 2
Mean (-) 1.96 -2 (-) 2264442 -11
Solvent Control 1 1.92 1972263
2 1.93 2081189
3 1.96 2247171
4 1.96 2260477
5 1.86 1677677
6 1.92 2020468
Mean 1.93 2043208
Control 1 1.86 1655914
2 1.77 1250824
3 1.88 1766814
4 1.82 1486098
5 1.79 1366572
6 1.85 1625216
Mean 1.83 1525240

Negative values = growth stimulation

Summary of analytical data

Measured concentrations and percentage of the geometric mean measured test item concentration at start of the exposure (0 hours) and end of the exposure (72 hours).

Sampling date 18.07.2016 21.07.2016
0 hours 72 hours
Start of the exposure End of the exposure
Concentration [µg/L] Meas.conc. [µg/L] % Meas.conc. [µg/L] % Geometric mean
measured test item
concentration 2)
[µg/L]
1000 7681) 771) 40.5 4 176
500 413 83 9.83 2 63.7
250 239 96 4.7 2 33.5
125 108 86 < LOQ 0 12.7
62.5 45.61)  731)  < LOQ 0 8.27
Control < LOQ < LOQ
Solvent Control < LOQ < LOQ

Meas. Conc = Measured concentration of the test item (dilution and weighing factors taken into account) = Percent of the nominal concentration of the test item

LOQ = Limit of Quantification (3 pg/L test item)

1) = Reanalyzed on 2016-07-19, mean value of 3 replicates with 2 injections each

2) = values < LOQ were taken with % LOQ (1.5 pg/L) into account

Validity Criteria

The study meets the validity criteria of the guideline:

Validity Criteria Required  Control Solvent control
Increase of the cell growth in the control cultures

Exponentially ± 16-fold corresponding to a specific growth rate of

0.92 day -1

243-fold (specific growth rate 1.83 day-1)


325-fold
(specific 1.93 day
 -1 )

Mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures  ≤ 35 % 27.10% 24.90%
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures ≤ 7 % 2.36% 1.88%
Validity criteria fulfilled:
yes
Conclusions:
The derived effect concentrations were: 72h-ErC50 (Pseudokirchneriella subcapitata, growth rate) = 166 µg/L, 72h-ErC10 (Pseudokirchneriella subcapitata, growth rate) = 45.5 µg/L; 72h-NOErC (Pseudokirchneriella subcapitata, growth rate) = 12.7 µg/L; 72h-EbC50 (Pseudokirchneriella subcapitata, biomass) = 50.1 µg/L, 72h-EbC10 (Pseudokirchneriella subcapitata, biomass) = 18.8 µg/L and 72h-NOEbC (Pseudokirchneriella subcapitata, biomass) = 12.7 µg/L.
Executive summary:

The toxicity of the test substance Phytosphingosine to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to test guideline OECD 201 (adopted 2011) and in compliance with GLP. The aim of the study was the determination of NOEC, LOEC, EC10, EC20 and EC50-values of the growth rate and the yield of biomass over a period of 72 hours in natural river water.

The study was conducted under static conditions with an initial cell density of 6305 cells/mL. For the preparation of the test concentrations appropriate stock solutions for each test item concentration were prepared in DMF (solvent). Five concentration levels were tested in a geometrical series with a dilution factor of 2: 62.5 - 125 - 250 - 500 - 1000 µg/L. These concentration levels correspond to the geometric mean measured test item concentrations of 8.27 - 12.7 - 33.5 - 63.7 - 176 µg/L. Concentration of the solvent DMF in each concentration level was 0.05 mL/L.

Three replicates were tested for each test item concentration and six replicates for the solvent control and 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 Phytosphingosine in all test concentrations and the controls were analytically verified by LC-MS/MS at the start (0 hours) and at the end (72 hours) of the exposure. The measured concentrations of Phytosphingosine in the fresh media (0 h) were in the range of 45.6 to 768 µg test item/L. The measured concentrations in the old media (72 h) were in the range of < LOQ (1.5 µg/L) to 40.5 µg test item/L.

During the 72 hours exposure period, inhibition of growth (r) and inhibition of yield (b) were observed in a dose-response relationship. No adverse effects were observed in the two lowest test concentration, i.e. 8.27 and 12.7 µg/L. The derived effect concentrations for growth inhibition were: 72h-ErC50 = 166 µg/L, 72h-ErC10 = 45.5 µg/L, and 72h-NOErC = 12.7 µg/L. The derived effect concentrations for yield of biomasse were: 72h-EbC50 = 50.1 µg/L, 72h-EbC10 = 18.8 µg/L, and 72h-NOEbC = 12.7 µg/L. Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities. All effect values were given based on the geometric mean measured test item concentrations.

The validity criteria of the guideline were fulfilled and results considered reliable and adequate for the aquatic hazard assessment.

Description of key information

72h-ErC50 (Pseudokirchneriella subcapitata, growth rate) = 166 µg/L (mean measured, OECD 201, 2016)

72h-ErC10 (Pseudokirchneriella subcapitata, growth rate) = 45.5 µg/L (mean measured, OECD 201, 2016)

72h-NOErC (Pseudokirchneriella subcapitata, growth rate) = 12.7 µg/L (mean measured, OECD 201, 2016)

Key value for chemical safety assessment

EC50 for freshwater algae:
166 µg/L
EC10 or NOEC for freshwater algae:
45.5 µg/L

Additional information

A reliable study was available investigating the toxicity of the Phytosphingosine to the unicellular freshwater green alga Pseudokirchneriella subcapitata according to test guideline OECD 201 (adopted 2011) and in compliance with GLP. The study was conducted under static conditions in natural river water and five concentration levels were tested in a geometrical series (62.5 - 125 - 250 - 500 - 1000 µg/L); controls and solvent controls were run in parallel. The concentrations of the test item Phytosphingosine in all test concentrations and the controls were analytically verified by LC-MS/MS.

During the 72h exposure period, inhibition of growth (r) and inhibition of yield (b) were observed in a dose-response relationship. No adverse effects were observed in the two lowest test concentration, i.e. 8.27 and 12.7 µg/L. The derived effect concentrations for growth inhibition were: 72h-ErC50 = 166 µg/L, 72h-ErC10 = 45.5 µg/L, and 72h-NOErC = 12.7 µg/L (based on geom. measured concentrations. The derived effect concentrations for yield of biomass were: 72h-EbC50 = 50.1 µg/L, 72h-EbC10 = 18.8 µg/L, and 72h-NOEbC = 12.7 µg/L (based on geom. measured concentrations). Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities.

The validity criteria of the guideline were fulfilled and results considered reliable and adequate for the aquatic hazard assessment.