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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:
2016-07-01 to 2016-07-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
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: 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. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15AB0305
- manufacture date: 2015-01-24
- Expiration date of the lot/batch: 2017-01-23 (retest date)
- Purity test date: 2015-09-22

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

OTHER SPECIFICS:
- purity: 99.7%
- appearance: white powder
- Water solubility: not available
Analytical monitoring:
yes
Details on sampling:
- Sampling method: samples were taken before the start of the test, after 24 hours and after 72 hours from each test medium and from the control. 1.0 mL of volume was taken. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. In addition, the filter containing undissolved residue was kept for possible analysis.
- Sample storage conditions before analysis: stored in a freezer until analysis. Additionally, reserve samples of 1.0 mL were taken for possible 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 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.
- 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
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1) 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.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2, according to OECD 201) 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:
yes
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
20-22°C
pH:
t=0h: 7.8 - 7.9
t=72h: 7.5-7.6
Dissolved oxygen:
not reported
Salinity:
not relevant
Conductivity:
not applicable
Nominal and measured concentrations:
- Nominal test concentrations: control and 100% of a SS prepared at 100 mg/L.
- Measured test concentrations:
* 0h (mg/L): n.a., < LOQ*
* 24h (mg/L): n.a., < LOQ*
* 72h (mg/L): n.a., < LOQ*

n.a. = not applicable
* The limit of quantification of the method was determined to be 0.01 mg/L

Details on test conditions:
TEST SYSTEM
- Test vessel: beaker
- Material, size, headspace, fill volume: 100 mL, all-glass, containing 50 mL of test solution
- Initial cell density: 1 x 10^4 cells/mL
- No. of vessels per concentration (replicates):
* 6 replicates each of the control and the highest concentration;
* 3 replicates of each lower concentrations;
* 1 extra replicate of the control and each test concentration for sampling purposes after 24 hours of exposure;
* 1 or 2 replicates of each concentration without algae.

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 were 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 (highest test concentration and control). Temperature was continuously measured in a control vessel. At the end of the final test microscopic observations were performed on the control and the highest test concentration to observe for any abnormal appearance of the algae.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- illumination: TLD-lamps with a light intensity within the range of 84 to 88 µE/m²/s.
- Adjustment of pH: no
- Light intensity and quality: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : growth inhibition
- Determination of cell concentrations: 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 (pathlength = 20mm).
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x10
- Range finding study: combined limit/range finding study was performed
- Test concentrations: 1.0, 10 and 100% of a SS prepared at 100 mg/L
- 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.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: At the nominal loading rate of 100 mg/L, no quantifiable amount of test substance were present in the test medium from the start of testing on
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: At the nominal loading rate of 100 mg/L, no quantifiable amount of test substance were present in the test medium from the start of testing on
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) > max. solubility of (of test item in test medium and loading rate initially prepared)
- 72-h EC10 (growth rate) > max. solubility of (of test item in test medium and loading rate initially prepared)
- 72-h EC10 (yield) < max. solubility of (of test item in test medium and loading rate initially prepared)
- 72-h NOEC (yield) = max. solubility of (of test item in test medium and loading rate initially prepared)
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 72h-ErC50 was 1.0 mg/L (95% confidence interval ranging from 0.97 to 1.0 mg/L)
- Other: the historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. The observed 72h-ErC50 for the algal culture tested corresponds with this range
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 highest test concentration compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Two-sample t-test Procedure, α=0.05, onesided, smaller).
No EC50-values could be calculated because the test item proved to be non-toxic (EC50 > maximum soluble concentration tested).
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 algal species Pseudokirchneriella subcapitata was performed with the test substance JNJ-42808415-AAA (T003422) according to the OECD guideline 201 (GLP conditions). No significant inhibition of growth rate was recorded at any of the concentrations of T003422 tested. The EC50 for growth rate inhibition (72-h ErC50) and the EC50 for yield inhibition (72-h EyC50) were beyond the range tested, i.e. exceeded the maximum soluble concentration of the test item in test medium at a loading rate of 100 mg/L. The 72-h NOEC for growth rate inhibition was determined to be at the solubility limit of the test item in test medium (NOELr = 100 mg/L). The 72h-NOEC for yield inhibition could not be determined. The results of the test can be considered reliable without restrictions.

Description of key information

The study of Tobor-Kaplon (2017), investigating the acute toxicity of T003422 to algae according to OECD guideline 201, was considered as the key study for endpoint coverage. The EC50 values (growth rate inhibition 72h EC50 and yield inhibition 72h EC50) of the test item were beyond the range tested, i.e. exceeded the maximum soluble concentration of the test item in test medium at a loading rate of 100 mg/L. The 72-h NOEC for growth rate inhibition was determined to be at the solubility limit of the test item in test medium (NOELR = 100 mg/L). The 72-h NOEC for yield inhibition could not be determined. The substance can be considered as not harmful to algae.

Key value for chemical safety assessment

Additional information

The measured concentration was not stable throughout the exposure duration, i.e. it decreased to 3.5% of initial within 24 hours of exposure, and could not be quantified anymore at the end of the test. Because the exposure concentrations could not be reliably determined, the effect parameters were expressed in terms of maximum solubility in test medium and the loading rates initially prepared.