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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: read-across from a guideline study
Justification for type of information:
The read across justification is presented in the Endpoint summary Aquatic toxicity. The accompanying files are also attached there.
Reason / purpose:
read-across source
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.16 mg/L
Basis for effect:
growth rate
Remarks on result:
other: read-across from Citronellyl butyrate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.083 mg/L
Basis for effect:
growth rate
Remarks on result:
other: read-across from Citronellyl butyrate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.043 mg/L
Basis for effect:
growth rate
Remarks on result:
other: read-across from Citronellyl butyrate
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 10 2016 - July 27 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The information is used for read across to Geranyl Isobutyrate MCS.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006, corrected 2011
Deviations:
yes
Remarks:
Composition of test medium was modified, see 'Details on test conditions'.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures
Version / remarks:
2000
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Remarks:
Swiss Federal Office of Public Health. Consumer protection directorate. Notification authority of chemicals. CH-3003 (d.d. 16 December 2015)
Specific details on test material used for the study:
Water solubility: 1.63 mg/L (at 20°C and pH 8.89)
Relative density: 0.8804
Log Pow: 6.20
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control, Undiluted Filtrate with a loading rate of 100 mg/L, Diluted filtrates at dilutions 1:10, 1:4.6, 1:2.2; at all sampling dates. The samples of the lowest test concentration (dilution 1:22) were not analyzed since these concentrations were below the 48-hour NOEC determined in this test and were therefore not relevant for the interpretation of the biological results.
- Sampling method: Duplicate samples without algae were taken from the test media of all test concentrations and the control at the start of the test and duplicate stability samples with algae were taken, after 24 and 48 hours and at the end of the test (72 hours). The analytical stability samples (9 mL sample volume) at 24 and 48 hours could not be taken from the test vessels itself, as the principle of a closed system is, that the test vessels have to remain completely filled with test medium during the entire test period. Therefore, for each of the samplings at 24 and 48 hours, a set of additional flasks containing the corresponding test medium (with algae) was incubated under conditions identical to the test. For each sampling date one set was harvested and then disposed. Immediately after sampling, acetonitrile (6 mL acetonitrile and 9 mL sample volume) was added to each sample to stabilize the latter during the storage period.
- Sample storage conditions before analysis: all samples were deep-frozen (at about -20 °C)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: At the start of the test (Day 0) the highest test concentration (undiluted filtrate) was prepared by dosing 268 μL of the test item in 2350 mL test water. This volume is equivalent to a loading rate of 100 mg/L, considering the relative density of the test item of 0.88. The test item was mixed into test water by intensive stirring for three hours in a closed stirring vessel to dissolve a maximum of test item in test water. After stirring, the emulsion was filtered through a 0.45 μm membrane filter (Whatman, NC45). As a pre-caution,the filter was pre-conditioned with 200 mL filtrate to avoid losses of dissolved test item by adsorption on the filter material. The undiluted filtrate was used as highest test concentration. It was further diluted with test water to prepare the test media with the lower test concentrations.
- Concentrations: Undiluted filtrate of the test item emulsion with a loading rate of 100 mg/L and
the dilutions 1:2.2, 1:4.6, 1:10, and 1:22.
- Controls: Test water without test item.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Strain No. 61.81 SAG
- Source: Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany). The algae were cultivated at the laboratories of the Test Facility.
- Age of inoculum at test initiation: 4 days
- Method of cultivation: An inoculum culture was set up four days before the start of the exposure. The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.15 mmol/L (= 15 mg/L as CaCO3)
Test temperature:
22.5-22.6 °C
pH:
7.9-8.0
Nominal and measured concentrations:
Nominal: Control, Undiluted filtrate with a loading rate of 100 mg/L, and dilutions of filtrate at 1:2.2, 1:4.6, 1:10, and 1:22.
Mean measured concentrations: Undiluted filtrate - 0.16 mg/L; dilution 1:2.2 - 0.063 mg/L; dilution 1:4.6 - 0.052 mg/L; dilution 1.10 - 0.043 mg/L (Mean measured concentration calculated as the geometric mean of the concentrations measured at all sampling dates (0, 24, 48, and 72 hours); see Table in "Any other information on results incl. tables" for measured concentrations at each time point.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks nearly completely filled (without headspace) with 60 mL test meadium and tightly sealed with glass stoppers (closed system).
- Aeration: no
- Initial cells density: 5000 cells/mL (nominal), 1.19 x 10^4 relative fluorescence units
- Control end cells density: 119 x 10^4 relative fluorescence units.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Standard medium (AAP according to OECD 201), modified according to ISO 14442.
- Detailed composition if non-standard medium was used: Concentration of NaHCO3 was increased to 250 mg/L and 6 mmol/L HEPES buffer (corresponding to 1430 mg/L) were added to keep pH of the test media as constant as possible.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile purified water
- Culture medium different from test medium: not specified
- Intervals of water quality measurement: pH was measured and recorded in each treatment at the start and end of the test. The temperature in the incubator was monitored and recorded continuously.The appearance of the test media was also visually controlled and recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous illumination by LED light installed above the test flasks.
- Light intensity: light intensity at the level of the test solutions was approximately 67 μE s^-1m^-2 (range: 64 to 69 μE s^-1m^-2, measured at nine places in the experimental area).
- The flasks were incubated in a temperature controlled orbital shaker and were positioned randomly and repositioned daily.

EFFECT PARAMETERS MEASURED
- The algal biomass was determined by fluorescence measurement at 24, 48 and 72 hours after exposure.
- A small volume (2 x 100 μL) of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced.
- The algal biomass in the samples was determined by fluorescence measurement (SpectraMax I3x, Molecular Devices Ltd, Wokingham Berkshire/UK). The measurements were performed at least in duplicate at an excitation of 440 nm and emission of 680 nm.
- At the end of the test, a sample was taken from the control and from the highest test concentration (undiluted filtrate) to determine a potential influence of the test item on the algal cells. The shape and size of the algal cells were visually inspected.

TEST CONCENTRATIONS
- Following the results of a stirring pre-experiment, the stirring/filtration method with a stirring period of three hours (closed stirring vessel) was applied for the preparation of test media.
- Range finding study test concentrations: Control, dilution 1:20, dilution 1:5, undiluted filtrate at loading rate 100 mg/L
- Results used to determine the conditions for the definitive study: yes.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (performed July 2016)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.16 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.083 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval 0.076-0.088
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.043 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none observed
- Other observations: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: The 72-hour EC50 for growth rate in the reference test was 1.0 mg/L (July 2016) and showed that the sensitivity of the test system was within the range recommended by the guideline (72-hour EC50 for the growth rate 0.92-1.46 mg/L).
Reported statistics and error estimates:
The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and the 95% confidence intervals were calculated as far as possible by Probit Analysis using linear maximum likelihood regression.
For the determination of the LOEC and NOEC, the average growth rate at the test concentrations were compared to the control values by the Williams t-test. Statistical analysis was performed using ToxRat Professional®.

Measured concentrations at each time point:

Sampling Day/ Age of Sample

[d/h]

Dilution Factor

of Filtrate of

Loading Rate

100 mg Test Item /L

Measured

Concentration

of Test Item

x

[mg/L]

Sample

Preparation

Factor

F

Determined

Concentration

of Test Item

c

[mg/L]

0/0 (fresh)

Control

1:10 Diluted Filtrate

1:4.6-Diluted Filtrate

1:2.2-Diluted Filtrate

Undiluted Filtrate

n.d.

0.0484

0.108

0.223

0.460

1.667

1.667

1.667

1.667

1.667

<LOQ

0.0807

0.180

0.372

0.767

1/24 (aged)

Control

1:10 Diluted Filtrate

1:4.6-Diluted Filtrate

1:2.2-Diluted Filtrate

Undiluted Filtrate

n.d.

n.d.

n.d.

n.d.

0.462

1.667

1.667

1.667

1.667

1.667

<LOQ

<LOQ

<LOQ

<LOQ

0.770

2/48 (fresh)

Control

1:10 Diluted Filtrate

1:4.6-Diluted Filtrate

1:2.2-Diluted Filtrate

Undiluted Filtrate

n.d.

n.d.

n.d.

n.d.

n.d.

1.667

1.667

1.667

1.667

1.667

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

3/72 (aged)

Control

1:10 Diluted Filtrate

1:4.6-Diluted Filtrate

1:2.2-Diluted Filtrate

Undiluted Filtrate

n.d.

n.d.

n.d.

n.d.

n.d.

1.667

1.667

1.667

1.667

1.667

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

LOQ: 0.069 mg/L

n.d.: not detected

n.a.: not applicable

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' section for details on validity criteria
Conclusions:
In the freshwater green algal species Pseudokirchneriella subcapitata the 72-h ErC50 and ErC10 values are >0.16 mg/L and 0.083 mg/L, respectively, based on mean measured concentrations of the test item. The NOEC is 0.043 mg/L.
Executive summary:

The toxicity to freshwater algae (Pseudokirchneriella subcapitata) was examined in a study according to OECD TG 201 and in compliance with GLP criteria. Algae, at a starting cell concentration of 5E+03 cell/mL (nominal) were exposed to Citronellyl butyrate as an undiluted filtrate with a loading rate of 100 mg/L and to dilutions of this filtrate at 1:22, 1:10, 1:4.6 and 1:2.2. Test substance analysis was performed on the dilutions 1:10, 1:4.6 and 1:2.2 and on the undiluted filtrate. The respective measured concentrations were determined to be 0.043, 0.052, 0.063 and 0.16 mg/L. These measured concentrations were calculated as the geometric mean of the concentrations measured at all sampling times 0, 24, 48, and 72 hours. After 72 hours exposure, no statistically significant inhibitory effect on the growth rate of algae was seen up to and including the test item concentration of 0.043 mg/L. At the test concentrations of 0.052, 0.063 and 0.16 mg/L, inhibition of growth rates were 3.5%, 5.7% and 34.6%, respectively. Based on these findings, the 72-h ErC50 and ErC10 values are determined to be >0.16 and 0.083 mg/L, respectively (based on mean measured concentrations). The NOErC is 0.043 mg/L. The validity criteria were met and the study considered to be reliable without restrictions.

Description of key information

The toxicity to aquatic algae is assessed based on read-across from the close structural analogue Citronellyl butyrate (CAS# 141 -16 -2).

For Citronellyl butyrate, the toxicity to freshwater algae (Pseudokirchneriella subcapitata) was examined in a study according to OECD TG 201 and in compliance with GLP criteria (Symrise, 2016). Algae, at a starting cell concentration of 5E+03 cell/mL (nominal), were exposed to Citronellyl butyrate as an undiluted filtrate with a loading rate of 100 mg/L and to dilutions of this filtrate at 1:22, 1:10, 1:4.6 and 1:2.2. Test substance analysis was performed on the dilutions 1:10, 1:4.6 and 1:2.2 and on the undiluted filtrate. The respective measured concentrations were determined to be 0.043, 0.052, 0.063 and 0.16 mg/L. These measured concentrations were calculated as the geometric mean of the concentrations measured at all sampling times 0, 24, 48, and 72 hours. After 72 hours exposure, no statistically significant inhibitory effect on the growth rate of algae was seen up to and including the test item concentration of 0.043 mg/L. At the test concentrations of 0.052, 0.063 and 0.16 mg/L, inhibition of growth rates were 3.5%, 5.7% and 34.6%, respectively. Based on these findings, the 72-h ErC50 and ErC10 values are determined to be >0.16 and 0.083 mg/L, respectively (based on mean measured concentrations). The NOErC is 0.043 mg/L. The validity criteria were met and the study considered to be reliable without restrictions.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
0.083 mg/L

Additional information

See CSR section 7.1.