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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:
March 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
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
FRESKOMENTHE
Lot No. VE00426966
Purity 99.2%
Expiry Date 19 February, 2018
Analytical monitoring:
yes
Details on sampling:
All concentration levels and the control were analytically verified via GC-FID after 0 hours (start of exposure) and 72 hours (end of the exposure).
Separate replicates for the test item analysis after 0 hours and 72 hours were prepared with algae at the start of the exposure and incubated under test conditions until analysis.
Samples from additional time points (24 and 48 hours) were sampled and stored. However, these samples were not analysed.
Vehicle:
no
Details on test solutions:
Preparation of Stock Solution - A stock solution with a nominal concentration of 100 mg test item/L was freshly prepared with dilution water (algal medium). For preparation the density of the test item was taken into account. The stock solution was stirred for 25 minutes (1100 rpm, room temperature) with a magnetic stirrer until the test item was completely dissolved.

Exposure Test Concentrations - Six concentration levels were tested in a geometrical series with a dilution factor of 3.16 (square root of ten):
0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L. The concentration levels are based on the results of a preliminary range finding test.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test Organism - Pseudokirchneriella subcapitata HINDÄK, SAG 61.81;

Source - Sammlung von Algenkulturen (SAG) Pflanzenphysiologisches Institut der Universität Göttingen Nikolausberger Weg 18, D-37073 Göttingen;

Culturing at Test Facility - Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 microE/m2/s for 24 hours per day;

Culture Medium - Nutrient medium Z according to Luettge et al. (1994).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
Minimum Temp : 21.0 °C
Maximum Temp : 23.0 °C
Mean Temp : 22.0°C
pH:
pH 7.92 - 9.37
Nominal and measured concentrations:
The measured concentrations of FRESKOMENTHE in the fresh media (0 h) were in the range of 90 to 115 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 92 to 114 % of the nominal values of all concentrations, representing 94.4 to 101.7% of the measured concentration at day 0.
Details on test conditions:
Replicates - Control - 6 replicates, Treatment levels - 3 replicates. Separate replicates for each measuring time were prepared.

Test Vessels - Sterile headspace flasks (volume 59 mL) with aluminium tops with PTFE seals.

Initial Cell Density - 6305 cells/mL

Incubation - The flasks were positioned randomly and repositioned daily; Temperature - Nominal range: 21 - 24 °C, controlled at ± 2 °C; Agitation - Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm; Light Intensity - Approximately 4440 to 8880 lux, corresponding to 60 to 120 microE/m2/s; Light Regime - Continuous and Within ± 15 % over incubation area;

Test Volume - 59 mL
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
30.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95%CI 29.0 - 32.3 mg/L
Remarks:
Test Item stable over test duration (Nominal = Measured)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
9.78 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 7.87 - 12.2 mg/L
Remarks:
Test Item stable over test duration (Nominal = Measured)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Test Item stable over test duration (Nominal = Measured)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
11.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95% CI 9.94 - 14.0 mg/L
Remarks:
Test Item stable over test duration (Nominal = Measured)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.37 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
Yield
Remarks on result:
other: 95% CI 0.790 - 3.49 mg/L
Remarks:
Test Item stable over test duration (Nominal = Measured)
Results with reference substance (positive control):
The toxicity of potassium dichromate (Sigma, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2016-10-11 to 2016-10-14 (with headspace) and 2016- 10-10 to 2016-10-13 (without headspace), respectively. The reference item toxicity is in the valid range following test facility SOPs.

Closed test vessels without headspace were used to mimic testing a volatile test item and results were compared with the standard system in open vessels. The data of both set-ups are consistent. It is evident from these results that the use of a modified test system to reduce losses of the test item through volatility (completely filled and sealed vessels) is expected to give results consistent with that obtained in a conventional system. As such it can be considered that the results from the definitive test give a reliable result for the test item.

Results :
72 hr ErC50 with headspace : 0.435 mg/L (valid range 0.782 +/- 0.534 mg/L)
72 hr ErC50 without headspace : 0.811 mg/L (valid range 0.782 +/- 0.534 mg/L)

72 hr EyC50 with headspace : 0.230 mg/L (valid range 0.410 +/- 0.288 mg/L)
72 hr EyC50 without headspace : 0.387 mg/L (valid range 0.410 +/- 0.288 mg/L)


Validity Criteria :

The study meets all the validity criteria required by the test guideline -

1) Increase of cell growth in Control cultures : 132 fold (criteria > 16 -fold);

2) Mean coeff of var for section-by-section specific growth rates (Days 0 -1, 1 -2 and 2 -3) in the Control cultures : 16.6% (criteria < = 35%);

3) Coeff of var of average growth rates during the whole test period in replicate Control cultures : 1.11% (criteria < = 7%).

Validity criteria fulfilled:
yes
Conclusions:
The specific end-points determined in this study with respect to inhibition of algae exposed to FRESKOMENTHE :

72 hr Growth Rate :
ErC50 = 30.6 mg/L (95% CI 29.0 - 32.3 mg/L)
ErC10 = 9.78 mg/L (95% CI 7.87 - 12.2 mg/L)
NOErC = 3.16 mg/L

72 hr Yield :
EyC50 = 11.7 mg/L (95% CI 9.94 - 14.0 mg/L)
EyC10 = 1.37 mg/L (95% CI 0.790 - 3.49 mg/L)
NOEyC < 0.316 mg/L
Executive summary:

The specific end-points determined in this study with respect to inhibition of algae exposed to FRESKOMENTHE :

72 hr Growth Rate :

ErC50 = 30.6 mg/L (95% CI 29.0 - 32.3 mg/L)

ErC10 = 9.78 mg/L (95% CI 7.87 - 12.2 mg/L)

NOErC = 3.16 mg/L

72 hr Yield :

EyC50 = 11.7 mg/L (95% CI 9.94 - 14.0 mg/L)

EyC10 = 1-37 mg/L (95% CI 0.790 - 3.49 mg/L)

NOEyC < 0.316 mg/L

Description of key information

The specific end-points determined in this study with respect to inhibition of algae exposed to FRESKOMENTHE :

72 hr Growth Rate :

ErC50 = 30.6 mg/L (95% CI 29.0 - 32.3 mg/L)

ErC10 = 9.78 mg/L (95% CI 7.87 - 12.2 mg/L)

NOErC = 3.16 mg/L

72 hr Yield :

EyC50 = 11.7 mg/L (95% CI 9.94 - 14.0 mg/L)

EyC10 = 1.37 mg/L (95% CI 0.790 - 3.49 mg/L)

NOEyC < 0.316 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
30.6 mg/L
EC10 or NOEC for freshwater algae:
9.78 mg/L

Additional information