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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:
2018/11/05-2018/11/08
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:
yes
Remarks:
please refer to "any other informations on materials and methods"
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, 55116 Mainz (15.05.2018)
Specific details on test material used for the study:
The test item was stored in the test facility in a closed vessel at room temperature (20 ± 5°C).
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 32; 10; 3.2; 1; 0.32; 0.1 mg/L nominal
- Sampling method: The content of the test item in the test solutions was measured at the beginning of the test and every 24 hours.
- Sample preparation:
Sample Preparation for treatment 32 mg/L (twofold enrichment):
20 mL of the sample was mixed with 3 g NaCl, then the solution was extracted two times with the solvent dichloromethane (2 x 4 mL) and the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane after addition of 500 μL ISTD stock solution (1000 mg/L in dichloromethane) and the solution was measured via GC/FID. Twofold enrichment was achieved during extraction.
Sample Preparation for treatment 3.2 – 10 mg/L (fivefold enrichment):
50 mL of a solution in media was mixed with 3 g NaCl, then the solution was extracted two times with the solvent dichloromethane (2 x 4 mL) and the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane after addition of 500 μL ISTD stock solution (1000 mg/L in dichloromethane) and the solution was measured via GC/FID. Fivefold enrichment was achieved during extraction.
Sample Preparation for Control, and treatment 0.1 mg/L - 1 mg/L mg/L (tenfold enrichment):
100 mL of a solution in media was mixed with 6 g NaCl, then the solution was extracted two times with the solvent dichloromethane (2 x 4 mL) and the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane after addition of 500 μL ISTD stock solution (1000 mg/L in dichloromethane) and the solution was measured via GC/FID. Tenfold enrichment will be achieved during extraction.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution containing 32.0 mg/L test item in buffered algal medium (demineralised water enriched with minerals but without algae) was prepared. This was done by mixing 39.5 μL/L test item (based on a density of 0.81 g/mL, given by the sponsor) with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae). To guarantee complete dissolution, the solution was treated in an ultrasonic bath for 1 hour followed by shaking vigorously for 1 hour. Non-GLP pre-tests confirmed that this approach will result in a complete dissolution. The lower treatments were prepared by dilution of this stock solution with algal medium.
- Controls: Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test (performed under GLP conditions)
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: (SAG Strain Number) 86.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen)
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared

ACCLIMATION
- Acclimation period: For the pre-culture an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours under test conditions (5000 lux, 22.0 – 23.2 °C). Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of culture was determined.
- Culturing media and conditions (same as test or not): The algal medium corresponds to the OECD TG 201 medium, nutrient medium (demineralised water enriched with minerals):
Algal medium:
Stock solution I 10.0 mL (NH4Cl 1500 mg, MgCl2*6H2O 1200 mg, CaCl2*2H2O 1800 mg, MgSO4*7H2O 1500 mg, KH2PO4 160 mg, H2O deionised ad 1000 mL);
Stock solution II 1.0 mL (FeCl3*6H2O 64 mg, Na2EDTA*2H2O 100 mg, H2O deionised ad 1000 mL);
Stock solution III 1.0 mL (H3BO3 185 mg, MnCl2*4H2O 415 mg, ZnCl2 3 mg, CoCl2*6H2O 1.5 mg, CuCl2*2H2O 0.01 mg, Na2MoO4*2H2O 7 mg, H2O deionised ad 1000 mL);
Stock solution IV 1.0 mL (NaHCO3 50 g, H2O deionised ad 1000 mL);
H2O deionised ad 1000 mL
All solutions were sterilized before use. Stock solutions I and III and the deionised water were sterilized using an autoclave. Stock solutions II and IV were sterilized using filtration.To avoid a strong pH increase and CO2 shortage in the closed system, additional 6 mL/L of Stock Solution IV (sodium hydrogen carbonate solution (50 g/L)) was added and the pH was adjusted to pH 7.0.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
At the beginning of the experiment and every 24 hours, the content of the test item in the test solution was analytically determined
Test temperature:
22.8 – 24.1 °C
pH:
7.0-9.9, refer to table 1
Nominal and measured concentrations:
please refer to table 2
Details on test conditions:
TEST SYSTEM
- Test vessel: glass vessel
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, total volume 65 mL
- Initial cells density: 2.3 *10E3 cells/mL
- Control end cells density: please refer to table 3
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, corresponding to OECD TG 201 medium

OTHER TEST CONDITIONS
- Sterile test conditions: n/a, solutions were sterilized
- Photoperiod: continuous lighting
- Light intensity and quality: 5000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 3.2
- Range finding study: The concentrations to be tested are based on non GLP pre-tests
- Test concentrations: 32 / 10 / 3.2 / 1 / 0.32 / 0.1 mg/L nominal concentration
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
17.91 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
22.6 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
19.25 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
23.34 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
7.93 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
14.34 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
23.34 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
7.93 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Already at 24 hours (besides for the highest concentration) and at 48 h and 72 h of the test no test item could be detected for any test concentration. This observation can only be explained by the fact that the measurable test item concentration is biased by the presence of the test organism (adsorption or ingestion onto the algae cells). Thus, the test item was present but not measurable in the test solutions.
Results with reference substance (positive control):
- Results with reference substance valid? yes, in a seperate reference study (201803R301), under GLP conditions
- EC50: 0.79 mg/L growth rate and 0.39 mg/L yield (within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L))
Reported statistics and error estimates:
Calculation of results was performed with the help of validated software (Microsoft Excel ®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.2.1

table 3: Cell Number/mL

Nominal Concentration in mg/L Parameter Cell Number/mL
0 h 24 h 48 h 72 h
Blank control Mean 2260 9087 57460 234363
Blank control SD 0 2118 19108 41861
0.1 Mean 2260 12100 71920 335960
0.1 SD 0 468 20075 152902
0.32 Mean 2260 9213 49493 249507
0.32 SD 0 1848 18359 87274
1 Mean 2260 15720 114347 624327
1 SD 0 1448 17272 72776
3.2 Mean 2260 10340 54953 322893
3.2 SD 0 1645 10699 76228
10 Mean 2260 12140 50780 305147
10 SD 0 2747 10291 26477
32 Mean 2260 4767 5620 17080
32 SD 0 129 779 7036

SD = Standard Deviation

table 4: Growth rate µ, Yield

Nominal Concentration in mg/L Parameter Growth Rate (0 -72h) [day-1] Yield (0-72h) [Cell Concentration/mL]
Blank control Mean 1 .54 232103
SD 0.06 41861
0.1 Mean 1.64 333700
SD 0.18 152902
0.32 Mean 1.56 247247
SD 0.11 87274
1 Mean 1.87 622067
SD 0.04 72776
3.2 Mean 1.65 320633
SD 0.09 76228
10 Mean 1.63 302887
SD 0.03 26477
32 Mean 0.65 14820
SD 0.16 7036

SD = Standard Deviation

table 5: Inhibition values

Nominal concentration in mg/L % Inhibition
Growth Rate (0-72h) Yield (0-72h)
Blank control 0 0
0.1 -6.24 -43.77
0.32 -0.84 -6.52
1 -21.37 -168.01
3.2 -6.77 -38.14
10 -5.94 -30.50
32 57.82 93.61

negative inhibition values indicate a stimulation of algal growth compared to the blank control

Validity criteria fulfilled:
yes
Conclusions:
The following results for the test item hexanal were determined:
Growth rate:
72 h-NOEC = 7.93 mg/L
72 h-LOEC = 23.34 mg/L
72 h-EC10 = 19.25 mg/L
72 h-EC50 = 22.60 mg/L
Yield (biomass):
72 h-NOEC = 7.93 mg/L
72 h- LOEC = 23.34 mg/L
72 h-EC10 = 14.34 mg/L
72 h-EC50 = 17.91 mg/L
Executive summary:

A study was performed in order to evaluate the toxicity of hexanal towards Desmodesmus subspicatus according to OECD guideline 201 and Commission Regulation (EU) No. 2016/266 amending Regulation (EC) No. 440/2008. This freshwater green alga was chosen as a typical species of phytoplankton. One valid experiment was performed. The study was performed using 6 concentrations ranging from 0.1 to 32 mg/L (nominal). To avoid volatilization of the test item, the study was performed in closed vessels. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 ±1 hours with an electronic particle counter. Growth rate μ and the yield1 were determined from the cell number at the respective observation times. Significant inhibition of algal growth was observed only at the highest concentration 32 mg/L. In the lower concentrations a stimulation of algal growth was observed. The content of the test item in the test solutions was measured at the beginning of the test and every 24 hours. In the treatments 0.32 – 32 mg/L the measured concentrations at the beginning of the test, before addition of algal pre-culture, were in a range between 55 % and 79 % of the nominal concentration. In the lowest concentration 0.1 mg/L the measured concentration was below the LOQ. After 24 hours, test item could be clearly detected only in the highest concentration. No test item was detectable in the measurements after 48 hours and 72 hours. During validation, at the concentration 32 mg/L the test item was shown to be stable in test medium without algae up to 72 hours. However, already at 24 hours (besides for the highest concentration) and at 48 h and 72 h of the test no test item could be detected for any test concentration. This observation can only be explained by the fact that the measurable test item concentration is biased by the presence of the test organism (adsorption or ingestion onto the algae cells). Thus, the test item was present but not measurable in the test solutions. As the test substance was biologically available, the biological results are related to the measured concentrations at the beginning before addition of algal pre-culture. This procedure is in agreement with the OECD guideline No. 201. Because in the lowest concentrated treatment the measured concentration at the beginning was lower than the LOQ, this treatment was excluded from statistical evaluation. The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

The following results for the test item Hexanal were determined:

Growth rate:

72 h-NOEC = 7.93 mg/L

72 h-LOEC = 23.34 mg/L

72 h-EC10 = 19.25 mg/L

72 h-EC50 = 22.60 mg/L

Yield (biomass):

72 h-NOEC = 7.93 mg/L

72 h- LOEC = 23.34 mg/L

72 h-EC10 = 14.34 mg/L

72 h-EC50 = 17.91 mg/L

Description of key information

D. subspicatus, 72 h, OECD 201, GLP, EC50 = 22.6 mg/L, EC10 = 19.25 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
22.6 mg/L
EC10 or NOEC for freshwater algae:
19.25 mg/L

Additional information

A study was performed in order to evaluate the toxicity of hexanal towards Desmodesmus subspicatus according to OECD guideline 201 and Commission Regulation (EU) No. 2016/266 amending Regulation (EC) No. 440/2008. The study was performed under GLP conditions. This freshwater green alga was chosen as a typical species of phytoplankton. One valid experiment was performed. The study was performed using 6 concentrations ranging from 0.1 to 32 mg/L (nominal). To avoid volatilization of the test item, the study was performed in closed vessels. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 ±1 hours with an electronic particle counter. Growth rate μ and the yield1 were determined from the cell number at the respective observation times. Significant inhibition of algal growth was observed only at the highest concentration 32 mg/L. In the lower concentrations a stimulation of algal growth was observed. The content of the test item in the test solutions was measured at the beginning of the test and every 24 hours. In the treatments 0.32 – 32 mg/L the measured concentrations at the beginning of the test, before addition of algal pre-culture, were in a range between 55 % and 79 % of the nominal concentration. In the lowest concentration 0.1 mg/L the measured concentration was below the LOQ. After 24 hours, test item could be clearly detected only in the highest concentration. No test item was detectable in the measurements after 48 hours and 72 hours. During validation, at the concentration 32 mg/L the test item was shown to be stable in test medium without algae up to 72 hours. However, already at 24 hours (besides for the highest concentration) and at 48 h and 72 h of the test no test item could be detected for any test concentration. This observation can only be explained by the fact that the measurable test item concentration is biased by the presence of the test organism (adsorption or ingestion onto the algae cells). Thus, the test item was present but not measurable in the test solutions. As the test substance was biologically available, the biological results are related to the measured concentrations at the beginning before addition of algal pre-culture. This procedure is in agreement with the OECD guideline No. 201. Because in the lowest concentrated treatment the measured concentration at the beginning was lower than the LOQ, this treatment was excluded from statistical evaluation. The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

The following results for the test item Hexanal were determined:

Growth rate:

72 h-NOEC = 7.93 mg/L

72 h-LOEC = 23.34 mg/L

72 h-EC10 = 19.25 mg/L

72 h-EC50 = 22.60 mg/L

Yield (biomass):

72 h-NOEC = 7.93 mg/L

72 h- LOEC = 23.34 mg/L

72 h-EC10 = 14.34 mg/L

72 h-EC50 = 17.91 mg/L