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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:
2003-03-18 to 2003-11-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Nonanal
- Appearance: Clear liquid
- Lot No.: 2F4107
- Expiration date of the lot/batch: March 2003
- Storage condition of test material: room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Analytical samples (approximately 10 mL) were collected into 40 mL glass VOA vials that contained 2 mL of 0.01M H2SO4 and 0.5 mL methanol in HPLC water. Samples collected at the start of the toxicity test were collected from test solutions just prior to the addition of algae and distribution of the test solutions to sealed test vessels. Samples collected at the end of the toxicity test were centrifuged to remove algal cells from pooled replicate test vessels. Samples outside the calibration range were diluted into the range with 50/50 acetonitrile/HPLC water.

Analysis of the following samples was performed at 0 and 72 hours:
- test samples: 0 (control), 0.25, 0.50, 1.0, 2.0, 4.0, 8.0 mg/L nominal
- laboratory control samples (standards prepared in dilution water): 2.0 mg/L
- stability samples (sample incubtaed with test vessels, not inoculated with algae): 8.0 mg/L
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Algae
- Strain: Selenastrum capricornutum, UTEX 1648
- Source (laboratory, culture collection): The culture was originally procured from the Culture Collection of Algae at the University of Texas at Austin and delivered to T.R. Wilbury Laboratories on July 17, 2001.
- Age of inoculum (at test initiation): 8 days
- Method of cultivation: No data

ACCLIMATION
- Acclimation period: at least 14 days
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: No data
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable
Hardness:
No data
Test temperature:
24 ± 2 °C
pH:
Initial: 7.4 - 7.6
Final: 7.1 - 9.9
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
Test sample results:
Nominal test concentrations: 0, 0.25, 0.50, 1.0, 2.0, 4.0 and 8.0 mg/L
Initial measured concentrations: 0, 0.196, 0.453, 0.759, 1.47, 3.20 and 6.41 mg/L
Final measured concentrations after 72 h: 0, ND, <0.00440, 0.0187*, 0.0530*, 0.212* and 0.373* mg/L
* estimated value because the instrument response was less than the lowest standard

Laboratory Control Samples (2 replicates):
Nominal: 2mg/l
0 hour: 1.74, 1.81 mg/L
72 hour: 1.41, 1.48 mg/L

Stability Sample:
Nominal: 8mg/L
0 hour: 6.54 mg/L
72 hour: 5.14 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: closed (sealed with Teflon-lined caps)
- Material, size, headspace, fill volume: clear glass 40 mL vials were filled to capacity to eliminate any head space
- Aeration: No
- Initial cells density: approximately 10000 algal cells/mL
- Control end cells density: 222000 cells/mL after 72 h
- No. of vessels per concentration (replicates): 11
- No. of vessels per control (replicates): 20
- A stability sample, prepared with a nominal concentration of 8.0 mg/L but not inoculated with algae, was incubated among the test vessels during the 72 hour exposure.

GROWTH MEDIUM
- Standard medium used: yes (freshwater AAP medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Sterile deionised water used to formulate the freshwater AAP medium
- Total organic carbon: Not detected (ND)
- Particulate matter: No data
- Metals:
- Aluminium: 0.02 mg/L
- Arsenic: ND
- Boron: ND
- Cadmium: ND
- Calcium: ND
- Chromium: ND
- Cobalt: 0.0034 mg/L
- Copper: ND
- Iron: ND
- Lead: 0.001 mg/L
- Magnesium: ND
- Mercury: ND
- Nickel: ND
- Potassium: ND
- Silver: ND
- Sodium: ND
- Zinc: 0.010 mg/L
- Pesticides: No data
- Chlorine: No data
- Alkalinity: No data
- Ca/mg ratio: ND
- Conductivity:No data
- Culture medium different from test medium: No
- Intervals of water quality measurement: Full analysis of the water was performed twice per year. The temperature in a representative vessel of water incubated with the test vessels was recorded continuously during the test. The pH of test solutions was measured and recorded in the single solution of each concentration prior to its distribution to test vessels at the beginning of the test, and in all test vessels used for the determination of the number of algal cells/mL at the end of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: pH was adjusted to 7.5 ± 0.1
- Photoperiod: 24 hour light maintained with cool-white fluorescent lights
- Light intensity and quality: approximately 400 footcandles (approximately 52 to 54 μEin/m²sec)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The number of algal cells/mL in each test vessel and the occurrence of relative size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic examination with a hemocytometer. At 24, 48, and 72 hours, three treatment vessels and six control vessels were randomly selected and sacrificed (opened to the atmosphere) to allow daily determination of the number of algal cells/mL. The remaining two vessels at each concentration were used for the determination of nonanal concentration at the end of the test.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0, 0.01, 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: At the conclusion of the range-finding test, the percent of control growth was as follows; 0.01, 0.10, and 1.0 mg/L = >100%, 10 mg/L = 9%, and 100 mg/L = <3%. A definitive test was initiated with a control and six concentrations of nonanal (0.50, 1.0, 2.0, 3.8, 7.5, and 15 mg/L). The test was terminated after 48 hours and repeated because analytical recoveries in samples collected at the start of the test were unacceptably low.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4.5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 4.03 - 5.02 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.6 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95% CL 2.17 - 3.11 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.79 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL 1.36 - 2.37 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.759 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: growth rate, cell number & biomass
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: No effects (relative size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells) were noted during the test.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Insoluble material was not observed during the test.
- At the conclusion of the definitive toxicity test, a 0.5 mL aliquot of test media from each test vessel where growth was maximally inhibited (the 3.20 and 6.41 mg/L nonanal concentrations) was combined with 100 mL of fresh media in a 250-mL flask. The cultures were incubated under test conditions for 72 hours. During this period the number of algal cells increased from an initial concentration of 1,600 cells/mL to approximately 580,000 cells/mL at 3.20 mg/L and from 330 cells/mL to 198,000 cells/mL at 6.41 mg/L, indicating that the toxic effects were algistatic rather than algicidal.
- The measured concentrations at 0 and 72 hours for the test samples, stability sample and laboratory control samples are provided in the attached background material and in the "nominal and measured concentrations" section above. Initial measured concentrations of nonanal ranged from 74 to 91% of the nominal concentrations. Final measured concentrations were <1 to 5% of nominal concentrations (< 7 % initial). However, the stability sample incubated with test material but not inoculated with algae showed a final measured concentration that was 78% of the initial measured concentration indicating that the decrease observed in the test samples with algae was likey due to adsorption to the increasing algal biomass and/or degradation caused by the presence of algae. This is further supported by the laboratory control samples where the 72 hour measured concentration was 81-82% of the 0h measured concentration. Given that disappearance of the test substance from solution by adsorption to the algal biomass does not mean that it is lost from the test system and that the algae are still exposed to the test item and any degradation products throughout the exposure period, and given that the measured decline in the stability sample was only just below the allowed deviation of ± 20 %, it is considered appropriate to base the effect results on the initial measured concentrations.
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of nonanal to the freshwater alga, Selenastrum capricornutum was assessed according to OECD guideline 201.
Exposure of algae to nonanal for 72 hours resulted in a median effective concentration (EC50) of 4.50 mg/L when calculated using the average specific growth rate, 2.60 mg/L when calculated using the number of cells/mL, and 1.79 mg/L when calculated using the area under the growth curve. The 72 hour no observed effect concentration (NOEC) is 0.759 mg/L nonanal when determined using the number of cells/mL, the average specific growth rate, or the area under the growth curve.
At the conclusion of the definitive toxicity test, a 0.5 mL aliquot of test media from each test vessel where growth was maximally inhibited (the 3.20 and 6.41 mg/L nonanal concentrations) was combined with fresh media. These cultures were incubated under test conditions for 72 hours. During this period the number of algal cells increased from an initial concentration of 1,600 cells/mL to approximately 580,000 cells/mL at 3.20 mg/L and from 330 cells/mL to 198,000 cells/mL at 6.41 mg/L, indicating that the toxic effects were algistatic rather than algicidal.

The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v2.0, OECD 201 Guideline). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. Thus the 72-h EC50 and NOEC based on growth rate are used for classification purposes, which were determined in this study to be 4.5 mg/L and 0.759 mg/L respectively.

Description of key information

The acute toxicity of nonanal to the freshwater alga, Selenastrum capricornutum was assessed according to OECD guideline 201. 

Key value for chemical safety assessment

EC50 for freshwater algae:
4.5 mg/L
EC10 or NOEC for freshwater algae:
0.759 mg/L

Additional information

The acute toxicity of nonanal to the freshwater alga, Selenastrum capricornutum was assessed according to OECD guideline 201. The test was performed under static conditions in sealed containers from which all air space had been removed to minimize the potential loss of test substance from test solutions to the atmosphere. Analysis of test samples at 0 and 72 hours showed a decline in measured concentrations, with the final measured concentrations being < 7% of the initial measured concentrations. However, the stability sample incubated with test material but not inoculated with algae showed a final measured concentration that was 78% of the initial measured concentration indicating that the decrease observed in the test samples with algae was likey due to adsorption to the increasing algal biomass and/or degradation caused by the presence of algae. This is further supported by the laboratory control samples where the 72 hour measured concentration was 81-82% of the 0h measured concentration. Given that disappearance of the test substance from solution by adsorption to the algal biomass does not mean that it is lost from the test system and that the algae are still exposed to the test item and any degradation products throughout the exposure period, and given that the measured decline in the stability sample was only just below the allowed deviation of ± 20 %, it is considered appropriate to base the effect results on the initial measured concentrations.Exposure of algae to nonanal for 72 hours resulted in a median effective concentration (EC50) of 4.50 mg/L when calculated using the average specific growth rate, 2.60 mg/L when calculated using the number of cells/mL, and 1.79 mg/L when calculated using the area under the growth curve. The 72 hour no observed effect concentration (NOEC) is 0.759 mg/L when determined using the number of cells/mL, the average specific growth rate, or the area under the growth curve. At the conclusion of the definitive toxicity test, a 0.5 mL aliquot of test media from each test vessel where growth was maximally inhibited (the 3.20 and 6.41 mg/L nonanal concentrations) was combined with fresh media. These cultures were incubated under test conditions for 72 hours. During this period the number of algal cells increased from an initial concentration of 1,600 cells/mL to approximately 580,000 cells/mL at 3.20 mg/L and from 330 cells/mL to 198,000 cells/mL at 6.41 mg/L, indicating that the toxic effects were algistatic rather than algicidal.

The preferred observational endpoint in this study is algal growth rate inhibition because it is not dependent on the test design (see Guidance on information requirements and chemical safety assessment Chapter R.7b: Endpoint specific guidance v1.1). Thus the 72 hr ErC₅₀ is used for classification purposes.