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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:
April 2, 2013 - April 5, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth 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)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control) 62.5, 125, 250, 500 and 1000 mg a.i./L.- Sampling method: To avoid incorrect TOC data (because of the test system) a special sampling method was used: Living cells increase the NPOC content of the solutions, therefore parallel with the flasks containing the living cells other flasks containing no living cells were run during the test. Analytical samples were taken from the flasks containing no living cells. Analytical samples were taken from the flasks containing no animals at the beginning and at end of the experiment. Three replicate were analyzed from each diluted analytical samples.- Sample storage conditions before analysis: The samples were analysed immediately after sampling or immediately after dilution of the samples.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances): Active ingredient content of the test substance (46.3 % in water solution) was taken into account in order to prepare the appropriate test solutions, therefore an amount of 2160 mg test item was dissolved in one litre vehicle (OECD medium) by mechanical dispersion in order to give the 1000 mg a.i./L test concentration. The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM- Strain: Pseudokirchneriella subcapitata- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Germany.- Method of cultivation: The pre-culture was intended to give an amount of alga suspension suitable for the inoculation of test cultures. The pre-culture was incubated under the conditions of the study in an aerated Algal Growth Medium and used when still exponentially growing (after an incubation period of 3 days). The cell count of above culture was determined by microscopic method and this cell suspension was diluted with Algal Growth Medium to 10E+07 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.2 – 22.6 °C
pH:
7.52 – 8.98
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 62.5, 125, 250, 500 and 1000 mg a.i./L.Measured concentrations: 0 (control), 75.4; 129.8; 258.7; 522.6 and 1046.5 mg a.i./L.
Details on test conditions:
TEST SYSTEM- Test vessel: Erlenmeyer flasks- Material, size, headspace, fill volume: 250 mL Erlenmeyer flasks, continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.- Initial cells density: 10E+04 algal cells per mL test medium- No. of vessels per concentration (replicates): Three replicates- No. of vessels per control (replicates): Six replicatesGROWTH MEDIUM- Standard medium used: yesTEST MEDIUM / WATER PARAMETERS: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.OTHER TEST CONDITIONS- Adjustment of pH: No- Photoperiod: continuously illuminated - Light intensity and quality: 8116 lux (equivalent to 110 μE/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm).EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The cell numbers were determined at 24, 48 and 72 hours after the treatment by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.TEST CONCENTRATIONS- Spacing factor for test concentrations: 2.0- Range finding study: Two concentration range-finding tests were conducted to determine the approximate toxicity of the test item so that appropriate test concentrations can be selected for use in the definitive test to get the EC50 value of the test item.- Test concentrations: 2 replicates each concentrations and 3 replicates in the control group.Test 1: 0, 0.1, 1, 10 and 100 mg a.i./L (static, 72 hours)Test 2: 0, 125, 250, 500 and 1000 mg a.i./L (static, 72 hours)- Results used to determine the conditions for the definitive study:Test 1: 0, 1.2, 1.0, 0.6, 1.1 % inhibition of growth respectivelyTest 2: 0, -0.1, 1.0, 0.5, 10.2% inhibition of growth respectively
Reference substance (positive control):
yes
Remarks:
(at least twice per year by CitoxLAB)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 046.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1565.35-5730.85
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
522.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 046.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 940.32 – 1470.63
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
522.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 046.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 1125.58 – 2283.88
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
522.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes- Observation of abnormalities (for algal test): There was no observed morphological deviations during the experiment.
Results with reference substance (positive control):
Potassium dichromate is tested by CitoxLAB at least twice a year to demonstrate satisfactory test conditions. The date of the last study (Study Code: 13/019-022AL) with the reference item Potassium dichromate is (Batch Number: 0769128): 05 - 08 February 2013.The 72h ErC 50 : 0.87 mg/L, (95 % confidence limits: 0.79 – 0.96 mg/L)The 72h EbC 50 : 0.48 mg/L, (95 % confidence limits: 0.44 – 0.53 mg/L)The 72h EyC 50 : 0.45 mg/L, (95 % confidence limits: 0.41 – 0.50 mg/L)
Reported statistics and error estimates:
The section-by-section specific growth rates in the control cultures were assessed. The inhibition of alga growth was determined from the biomass, the average specific growth rate and from the yield. Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software. The ErC50, EbC50 and EyC50 values of the test item were determined using Probit analysis by TOXSTAT software. Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software. For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

Influence of the test item on the Growth of Pseudokirchneriella subcapitata:

Parameter (0-72 h)

Growth rate (r) [mg/L]

Yield (y) [mg/L]

Biomass (b) [mg/L]

EC50

>1046.5

>1046.5

>1046.5

95 % conf. limits

1565.35 -5730.85

940.32 -1470.63

1125.58 -2283.88

NOEC

522.6

522.6

522.6

LOEC

1046.5

1046.5

1046.5

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, yield and areas were significantly different from that of the control group in the examined concentration of 1046.5 mg a.i./L (measured). The overall NOEC was determined as 522.6 mg a.i./L (measured); the overall LOEC was determined as 1046.5 mg a.i./L (measured).

As the analytically measured concentration deviated more than 20 per cent from the nominal in one case (80%), the biological results are based on the measured geometric mean concentrations.

Validity criteria fulfilled:
yes
Remarks:
(cell density in control increased by a factor of 70.33, mean coefficient of variation for section-by-section specific growth rates in control was 8.47 %, coefficient of variation of average specific growth rates in control was 0.86%)
Conclusions:
The EC50 of the test item in Algae (Pseudokirchneriella subpicata) at 72 h exposure period was greater that 1046.5 mg a.i./L and the NOEC was determined to be 522.6 mg a..i./L, both based on the growth rate.
Executive summary:

The Growth inhibition test on Algae (Pseudokirchneriella subpicata) was performed in accordance with EU Method C.3 and OECD Guideline 201. The effect of test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours in a static system. Based on the two performed preliminary range-finding tests, five test concentrations in a geometric series (factor 2.0) and one control were tested in the main experiment: 0 (control), 62.5, 125, 250, 500 and 1000 mg a.i./L corresponding to measured geometric mean concentrations of 0 (control) 75.4, 129.8, 258.7, 522.6 and 1046.5 mg a.i./L. mg/L. The test design included three replicates at test concentrations and six replicates for the untreated control. The EC50 were determined to be > 1046.5 mg a.i./L (measured, basis for effect: growth rate, yield and biomass). With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, yield and areas were significantly different from that of the control group in the examined concentration of 1046.5 mg a.i./L (measured). The overall NOEC was determined as 522.6 mg a.i./L (measured); the overall LOEC was determined as 1046.5 mg/L (measured).

Description of key information

Key study: Test method OECD 201. GLP study. 72h-EC50 and the 72-NOEC were determined to be > 1046.5 and 522.6 mg a.i./L respectively, both based on the growth rate.

Key value for chemical safety assessment

EC50 for freshwater algae:
1 046.5 mg/L
EC10 or NOEC for freshwater algae:
522.6 mg/L

Additional information

Key study: The Growth inhibition test on Algae (Pseudokirchneriella subpicata) was performed in accordance with EU Method C.3 and OECD Guideline 201. The effect of test item up to 1000 mg a.i./L was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours in a static system. The EC50 were determined to be > 1046.5 mg a.i./L (measured, basis for effect: growth rate, yield and biomass). The overall NOEC was determined as 522.6 mg a.i./L (measured); the overall LOEC was determined as 1046.5 mg/L (measured).