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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:
2016-09-30 to 2016-10-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16AB0081
- Expiration date of the lot/batch: 2018-01-11 (retest date)
- Purity test date: 2016-02-18 (certificate of analysis release date)
- Purity: 99.3% (chromatographic purity by GC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: <0.01 g/L in water

Analytical monitoring:
yes
Details on sampling:
- Sampling method: Single samples were taken before the start of the test, after 24h and after 72 hours from all test concentrations and from the control. A volume of 2.0 mL was taken. The filter used for preparation of the SS was kept for possible analysis of the residue. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. Reserve samples of 2.0 mL were taken from all test solutions for possible analysis.
- Sample storage conditions before analysis: stored in a freezer (< -15°C) until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in test medium. The resulting aqueous mixture was filtered through a 0.45 µm membrane filter (Whatman; RC55) to remove the undissolved fraction of the test item. The filter was pre-conditioned with a small volume of test solution that was discarded. The obtained Saturated Solution (SS) was used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/ml.
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All final test solutions were clear and colourless
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. M1 medium was used.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

ACCLIMATION
- Acclimation period: not relevant (except pre-culture 3 days before start of the test)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
21-23°C
pH:
t=0h: 7.9-8.0
t=72h: 7.8
Dissolved oxygen:
not reported
Salinity:
not applicable
Nominal and measured concentrations:
Range-finder;:
nominal test concetrations: 100% SS prepared at 100 mg/L
measured test concentration combined limit/range-finding test t = 0h: 0.737 mg/L
measured test concentration combined limit/range-finding test t = 72h: 0.581 mg/L


nominal test concentrations final test: 10, 18, 32, 56 and 100% SS prepared at 100 mg/L
measured test concentration final test t= 0 h: n.d., 0.0756, 0.135, 0.243, 0.246, 0.425, 0.772 mg/L
measured test concentration final test t= 24h: n.d., 0.0763, 0.131, 0.237, 0.244, 0.409, 0.730 mg/L
measured test concentration final test t= 72h: n.d., 0.0683, 0.121, 0.224, 0.212, 0.397, 0.396, 0.383 mg/L
geometric mean measured test concentrations final test: n.d., 0.073, 0.13, 0.23, 0.41, 0.61 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): capped vessels
- Material, size, headspace, fill volume: 100 mL all-glass flasks filled with 50 mL test solution
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no flow-through system applied
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: 10,000 cells/mL
- Control end cells density: 113.2 x 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis
- Detailed composition if non-standard medium was used:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2, according to the OECD 201 Guideline, formulated using Milli-RO water.
- Culture medium different from test medium: Yes (M1 versus M2). Three days before the start of the test the algal stock culture (culture in M1) was inoculated in the same culture medium (M2) used in the test. The culture was maintained under the same conditions as used in the test.
- Intervals of measurements: pH was measured at the beginning and at the end of the test. Temperature was continuously measured in a control vessel. At the end of the final test microscopic observations were performed on all test concentrations to observe for any abnormal appearance of the algae.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: none
- Photoperiod: continuous illumination
- Light intensity and quality: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm, using TLD-lamps.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] At the beginning, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe.
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x 1.8
- Range finding study
- Test concentrations: 1.00, 10 and 100 % of the SS
- 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:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 100% v/v saturated solution, equivalent to a loading rate of 100 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.42 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.23 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
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

- NOEC (growth rate): 0.23 T000625 mg/L (based on geometric mean measured concentration)
- EC10 (growth rate): 0.42 T000625 mg/L (95% C.L. 0.35-0.49 mg/L) (based on geometric mean measured concentration)
- EC50 (growth rate): > 0.61 T000625 mg/L (based on geometric mean measured concentration)

- NOEC (yield) = 0.13 mg T000625 /L (based on geometric mean measured concentration)
- EC10 (yield) = 0.098 mg T000625 /L (95% C.L. 0.055-0.14 mg/L) (based on geometric mean measured concentration)
- EC50 (yield) = 0.63 mg T000625 /L (95% C.L. 0.51-0.88 mg/L) (based on geometric mean measured concentration)
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 72-h EC50 (growth rate) = 0.39 mg/L
Reported statistics and error estimates:
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller) or inhibition of yield (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α=0.05, one-sided, smaller).
Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of ECx values was based on 3- parameters normal CDF (cumulative distribution function).
The calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).
Validity criteria fulfilled:
yes
Conclusions:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance T000625 according to the OECD guideline 201 (GLP conditions). Under the test conditions, the test item had a significant inhibitory effect on the growth of Pseudokirchneriella subcapitata at a TWA concentration of 0.41 mg/L and higher during the 72-hour test period.
The 72h-NOEC for growth rate inhibition was set at a TWA concentration of 0.23 mg/L based on biological relevance. The 72h-ErC10 was 0.42 mg/L with a 95% confidence interval ranging from 0.35 to 0.49 mg/L. The 72h-NOEC for yield inhibition was determined to be 0.13 mg/L based on the TWA concentration. The EC50 for growth rate inhibition (72h-ErC50) was beyond the range tested, i.e. exceeded a TWA concentration of 0.61 mg/L as measured in the saturated solution prepared at a loading rate of 100 mg/L. Therefore the 72h-ErL50 was > 100 mg/L. The EC50 for yield inhibition (72h-EbC50) based on TWA concentrations was 0.63 mg/L with a 95% confidence interval ranging from 0.51 to 0.88 mg/L.
The results of the test can be considered reliable without restrictions.

Description of key information

The study of Tobot-Kaplon (2016), investigating the acute toxicity of T000625 to algae according to OECD guideline 201, was considered as the key study for endpoint coverage. Under the test conditions, the test item had a significant inhibitory effect on the growth of Pseudokirchneriella subcabitata. The 72h-NOEC for growth rate inhibition was set at a TWA concentration of 0.23 mg/L based on biological relevance. The 72h-ErC10 was calculated to be 0.42 mg/L. The EC50 for growth rate inhibition (72h-ErC50) was beyond the range tested, i.e. exceeded a TWA concentration of 0.61 mg/L as measured in the saturated solution prepared at a loading rate of 100 mg/L. Therefore, the ErL50 was > 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
0.42 mg/L

Additional information