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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:
2018-03-02 to 2018-03-29
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
Qualifier:
according to
Guideline:
other: OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 20141015
- Expiration date of the lot/batch: 2019-01-31
- Purity test date: 2017-01-31
- Purity: 99.35%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature. Tightly closed container in a dry and well-ventilated place
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data
Analytical monitoring:
yes
Details on sampling:
- Sampling method: For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media at all test concentrations and the control at the start of the test (without algae) and at the end of the test (containing algae). For sampling at the end of the test, the test medium of the treatment replicates was pooled.
- Sample storage conditions before analysis: All samples were frozen (at -20 ± 5 °C) immediately after sampling and stored until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method
*Pre-experiment:
The choice of the closed system was based on a stirring pre-experiment (IES Study 20170459) which showed that the test item was volatile. In the stirring pre-experiment (IES Study 20170459) the test item proved to be stable over 72 hours in a closed system.
*Range-finder:
The highest test concentration (undiluted filtrate) was prepared by giving 99 µL of the test item to 1000 mL of test water using intense stirring for 3 hours at room temperature in the dark in a closed vessel. After stirring, the suspension was filtered through a 0.45 µm membrane filter (Whatman, NC45). The filter was saturated by 200 mL test medium and the negative pressure of the filtration unit was reduced to a minimum to avoid losses of the test item. The undiluted filtrate was used as highest test concentration. It was further used in a series of dilutions with test water to prepare the test media with the lower test concentrations.

*Main test:
Due to the low water solubility of the test item, a suspension with the loading rate of 100 mg/L was prepared at the start of the test. 99 µL of the test item were added into 1000 mL of test water using intense stirring for 3 hours at room temperature in the dark in a closed vessel. After stirring, the suspension was filtered through a 0.45 µm membrane filter (Whatman, NC45). The filter was saturated by 200 mL test medium and the negative pressure of the filtration unit was reduced to a minimum to avoid losses of the test item.
The undiluted filtrate was used in a series of dilution steps with test water to obtain the dilutions 1:3.2, 1:10, 1:32, 1:100 and 1:320. The test media were prepared just before the start of the test.

- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): All test media were clear solutions throughout the test period.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany).
- Method of cultivation: The algae were cultivated at IES Ltd Laboratories under standardized conditions according to the test guidelines. 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.

ACCLIMATION
- Acclimation period: not relevant
- Culturing media and conditions (same as test or not): same as the test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
15 mg CaCO3/L
Test temperature:
The test flasks were incubated in a temperature controlled orbital shaker (Multitron-Pro, Infors HT, Bottmingen/Switzerland) at a temperature of 23 °C.
pH:
The pH of the test media was in the range of 7.5 to 7.7 during the test period.
Dissolved oxygen:
not reported
Salinity:
not relevant
Conductivity:
not relevant
Nominal and measured concentrations:
Range-finder:
nominal test concentrations: 1:100, 1:10 and undilute filtrate
measured test concentration t= 0 h: 0.408, 4.04 and 39.6 mg/L
measured test concentration t= 72h: 0.341, 3.48, 4.12 (without algae), 3.49 (without algae, without light), 37.7 mg/L

Final test:
nominal test concentrations : 0, 1:100, 1:32, 1:10, 1:3.2
measured test concentration t= 0 h: measured test concentration t= 72h:
LOQ: 0.131 mg test item /L
Details on test conditions:
TEST SYSTEM
- Test vessel: As the test item is a volatile substance, glass stoppered Erlenmeyer flasks were used completely filled (without headspace) with about 60 mL of test medium and tightly sealed with glass stoppers (closed system)
- Initial cells density: 5,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The algae were cultivated and tested in synthetic test water (AAP Medium), prepared according to the test guidelines, but modified according to the International Standard ISO 14442 [2] as a closed test system was applied. Analytical grade salts were dissolved in sterile purified water to obtain the following final nominal concentrations:

Ingredients
Macro-Nutrients:
NaHCO3 250.0 mg/L
K2HPO4 1.044 mg/L
MgSO4 × 7 H2O 14.6 mg/L
MgCl2 × 6 H2O 12.16 mg/L
CaCl2 × 2 H2O 4.41 mg/L
NaNO3 25.5 mg/L
Trace Elements:
H3BO3 186.0 µg/L
MnCl2 × 4 H2O 415.0 µg/L
ZnCl2 3.27 µg/L
CoCl2 × 6 H2O 1.43 µg/L
CuCl2 × 2 H2O 0.012 µg/L
Na2MoO4 × 2 H2O 7.26 µg/L
FeCl3 × 6 H2O 160.0 µg/L
Na2EDTA × 2 H2O 300.0 µg/L
The concentration of NaHCO3 was increased by 235 mg/L (as carbon source for the algal growth) and 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) were added to keep the pH of the test media as constant as possible. The pH of the test water was adjusted to 7.5. The water hardness (calculated) of the test water was 0.15 mmol/L (= 15 mg/L as CaCO3).
- Culture medium different from test medium: no
- Intervals of water quality measurement: The light intensity was measured at the start of the test. The 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 during the exposure period.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: yes , the pH of the test water was adjusted to 7.5.
- Photoperiod: continuous illumination
- Light intensity and quality: continuously illuminated by LED light installed above the test flasks. The light intensity at the level of the test solutions was approximately 64 µE s-1 m-2 (range: 62 to 68 µE s-1 m-2, measured at nine places in the experimental area).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] The algal cell density in the pre-culture was determined using an electronic particle counter (Cell Counter CASY TT, OLS, Bremen/Germany).
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 10 and 100% of the undilute filtrate
- 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.:
2.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.L. 2.8 - 3.0 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.L. 1.3 - 1.7 mg/L
Details on results:
- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- 72-h EC50 (yield) = 1.7 mg /L (95% C.L. 1.61 - 1.74 mg/L)
- 72-h EC10 (yield) = 1.0 mg /L (95% C.L. 0.96 - 1.03 mg/L)
- 72-h NOEC (yield) = 0.34 mg /L
- 72-h NOEC (growth) = 0.34 mg /L
Results with reference substance (positive control):
- Results with reference substance valid? yes, the sensitivity of the test system was within the range recommended by the guideline (72-hour EC50 for the growth rate 0.9 1.5 mg/L).
- 72-h EC50 (growth rate) = 1.1 mg/L
Reported statistics and error estimates:
The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95 % confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression.
For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by the Williams t test or Welch t-test, where appropriate.
Statistical analysis was performed using ToxRat Professional®.
Validity criteria fulfilled:
yes
Conclusions:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed wit the test substance butyl benzoate according to the OECD guideline 201 (GLP conditions). It can be concluded that the test item had a significant inhibitory effect on the growth of Pseudokirchneriella subcapitata up to and including the test concentration of 0.34 mg/L, after the test period of 72 hours. The analytically confirmed nominal EC50 for growth rate inhibition (72h-ErC50) was 2.9 mg/L with a 95% confidence interval ranging from 2.8 to 3.0 mg/L. The results of the test can be considered reliable without restrictions.

Description of key information

The study of Dupont (2018), investigating the acute toxicity of the test substance to algae according to OECD guideline 201, was considered as the key study for endpoint coverage. The 72-h EC10 and 72-h EC50 for growth rate were determined to be 1.5 mg/L and 2.9 mg/L respectively. The substance can be considered as toxic to algae.

Key value for chemical safety assessment

EC50 for freshwater algae:
2.9 mg/L
EC10 or NOEC for freshwater algae:
1.5 mg/L

Additional information