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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:
03 July 2017 - 06 July 2017
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:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Storage condition: Controlled room temperature (15-25°C, below 70 RH%)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 50 % saturated solution (of 0.125 mg/L nominal loading rate WAF) and 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 mg/L nominal loading rates WAFs.
- Sampling method: Samples were taken in plastic tubes, acidified with pure nitric acid
(9 mL sample + 1 mL HNO3)
- Sample storage conditions before analysis:(stored under -20°C until analysis)
Vehicle:
no
Details on test solutions:
The test item is a multi constituent substance and poorly soluble in water therefore a test solution was prepared using a saturated solution method (water accommodated fraction, WAF) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
Saturated test item solutions [based on nominal loading rates of 0.125, 0.25, 0.5, 1.0 , 2.0 and 4.0 mg/L (WAFs)] were prepared individually by dispersing/dissolving the amount of test item into the test medium (OECD Medium) two days before the start of the test. These solutions were shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give appropriate nominal loading rates.
As the lowest concentration cannot be prepared individually with a sufficient precision, therefore it was prepared from a serial dilution of the next concentration level (0.125 mg/L WAF) to give the 50 % saturated solution of 0.125 mg/L nominal loading rate WAF (200.0 mL 0.125 mg/L WAF solution was diluted with OECD Medium up to 400 mL).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: same
Test type:
not specified
Water media type:
freshwater
Remarks:
Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
The test was started (0 hours) by inoculation of a biomass of approximately 10^4 algal cells per mL test medium.
Hardness:
not specified
Test temperature:
22.6 – 22.8 °C measured in the flask and between 21.9 and 23.2 °C measured within the climate chamber
pH:
7.68 – 8.81
Dissolved oxygen:
not specified
Salinity:
not specified
Conductivity:
not specified
Nominal and measured concentrations:
nominal: 50 % saturated solution (of 0.125 mg/L nominal loading rate WAF) and 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 mg/L nominal loading rates WAFs.
measured: <0.18 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: flask
- No. of vessels per concentration (replicates): three replicates per test concentration and six replicates in the control group
GROWTH MEDIUM
- Standard medium used: yes
- Light intensity and quality: 8373 lux (equivalent to ~113 μE/m2/s)

Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
ca. 0.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
ca. 0.52 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
ca. 0.56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
ca. 0.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
ca. 0.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
ca. 0.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
ca. 0.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
ca. 0.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
ca. 0.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
biomass
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: Thin cells could be observed at the two highest concentration levels of 2.0 and 4.0 mg/L nominal loading rate WAF during the whole experiment
Results with reference substance (positive control):
- Results with reference substance valid: yes
The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EyC 50: 0.54 mg/L, (95 % confidence limits: 0.50 – 0.59 mg/L)
The 72h EbC 50: 0.64 mg/L, (95 % confidence limits: 0.59 – 0.70 mg/L)
Reported statistics and error estimates:
see above
Validity criteria fulfilled:
not specified
Conclusions:
B331 72-hour EL50 (algae) = 0.90 mg/L
With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the examined concentration range of 0.5 – 4.0 mg/L nominal loading rate WAF, therefore the NOELR was determined as 0.25 mg/L nominal loading rate WAF and the LOELR was determined as 0.50 mg/L nominal loading rate WAF.

Description of key information

Pseudokirchneriella subcapitata, 72 hours:

EL50 (growth rate) = 0.90 mg/L

NOELR = 0.25 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
0.9 mg/L
EC10 or NOEC for freshwater algae:
0.25 mg/L

Additional information

An acute algal inhibition study was conducted (CiToxLAB Hungary, 2017) to assess the short-term effects of B331 on aquatic plants. The study was conducted according to OECD test guideline 201 and EC method C3, and in compliance with GLP. The test media was prepared as a water accomodated fraction (WAF) with nominal loading rates of (50% saturated solution at 0.125 mg/L loading), 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0; a concurrent vehicle control group (untreated dilution water) was used.

Analysis of the test solutions in all dose levels at each sampling timepoint revealed the test material concentration to be below the limit of quantitation of 0.18 mg/L (measured for copper concent by ICP-AES; LoQ = 0.016 mg Cu/L).

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the examined concentration range of 0.5 – 4.0 mg/L nominal loading rate WAF, therefore the NOELR was determined as 0.25 mg/L nominal loading rate WAF and the LOELR was determined as 0.50 mg/L nominal loading rate