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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:
30 Mar 2015-24 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2006/2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Purity/composition correction factor: No correction factor required
Test substance handling: No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: INCI: Oleth-3 Carboxylic acid
Solubility in water: Not soluble
Stability in water: Stable
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, glass wool containing undissolved residue was kept for possible analysis.
Frequency: at t=0 h, t=24 h and t=72 h
Volume : 2.0 ml
Storage : Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at an intermediate substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, reserve samples of 2.0 ml were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
Test substance was not completely soluble in test medium at the loading rates initially prepared.

Water Accommodated Fractions (WAFs) were prepared at individual loading rates ranging from 0.32 to 100 mg/L applying a 2-day stirring period. Thereafter, the solutions containing undissolved material were left to stabilise for a period of 1 hour. Subsequently, the aqueous WAFs were collected by siphoning thorough glass wool and used as test solutions. The final test solutions were all clear and colourless except the WAF prepared at 100 mg/L in the combined limit/range-finding test that was observed to be colourless and hazy.

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 1E04 cells/mL.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Pseudokirchneriella subcapitata
Strain: NIVA CHL 1
Source (laboratory, culture collection): In-house laboratory culture
Age of inoculum (at test initiation):4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1E04 cells/mL.
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 (60 to 120 µE/m²/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm. in a climate room at a temperature of 21-24°C.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
no
Test temperature:
between 22 and 23°C
pH:
between 7.9 and 8.1
Nominal and measured concentrations:
Nominal concentrations: WAFs prepared at 0.32, 1.0, 3.2, 10 and 32 mg/L
Measured concentrations: The measured concentrations were at the level of 95-234% of the initial during the test. Based on these results, the Time Weight Average concentrations were calculated: TWA were resp. 0.30, 0.84, 1.7, 1.9, 9.2 and 25 mg/L
Details on test conditions:
TEST SYSTEM
- Type: open
- Material, size, headspace, fill volume: 100 mL, normal headspace, 50 mL
- Aeration: no
- Initial cells density: 10000 cells/mL
- Control end cells density: 1037000 cells/mL
Replicates:
3 replicates of each test concentration.
6 replicates of the control.
1 replicate of the intermediate concentration without algae.
1 replicate of each test concentration without algae.
1 replicate of each test concentration for sampling purposes at 24 hour of exposure

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 64 to 83 µE.m-2.s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber at test start, spectrophotometer daily thereafter


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Test concentrations: control and WAFs prepared at 0.32, 1.0, 3.2, 10 and 32 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.96 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.57-1.4 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
1.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.2-2.3 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 4.1-6.5 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.3 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.74 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95% CL: 0.60-0.86 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.91 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95% CL: 0.78 - 1.0 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95% CL: 1.2-1.5 mg/L
Details on results:
Exponential growth in the control: Yes
Observations of abnormalities: No abnormalities observed
Unusual cell shape: no
Colour differences: no
Results with reference substance (positive control):
The EC50 for growth rate inhibition (72h-ERC50) was 1.3 mg/l with a 95% confidence interval ranging from 1.1 to 1.5 mg/l.
The EC50 for yield inhibition (72h-EYC50) was 0.72 mg/l with a 95% confidence interval ranging from 0.63 to 0.83 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 or inhibition of yield (yield: Williams Multiple Sequential t-test Procedure, 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 probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding TWA concentrations of the test substance.

The calculations were performed with ToxRat Professional v. 2.10.05 (ToxRat Solutions® GmbH, Germany).
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, the EC50 for growth rate inhibition (72h-ERC50) is 5.2 mg/l with a 95% confidence interval ranging from 4.1 to 6.5 mg/l. The EC10 for growth rate is 0.96 mg/L with a 95% confidence interval ranging from 0.6 to 1.4 mg/L

The EC50 for yield inhibition (72h-EYC50) was 1.4 mg/l with a 95% confidence interval ranging from 1.2 to 1.5 mg/l.

The 72h-NOEC for yield inhibition was 0.30 mg/l. The NOEC for growth rate inhibition could not be determined.
Executive summary:

Under the conditions of the present study with Pseudokirchneriella subcapitata, the EC50 for growth rate inhibition (72h-ERC50) is 5.2 mg/L with a 95% confidence interval ranging from 4.1 to 6.5 mg/L. The EC10 for growth rate is 0.96 mg/L with a 95% confidence interval ranging from 0.6 to 1.4 mg/L

Description of key information

72h-ErC50 = 5.2 mg/L (95% confidence interval 4.1 to 6.5 mg/L), 72h-ErC10 = 0.96 mg/L (95% confidence interval 0.6 to 1.4 mg/L) (Pseudokirchneriella subcapitata) (RL1, GLP, OECD TG 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
5.2 mg/L
EC10 or NOEC for freshwater algae:
0.96 mg/L

Additional information

In a 72 hour toxicity study according to OECD Guideline 201 (2011), the cultures of Pseudokirchneriella subcapitata were exposed to Polyoxyethylene(1 to 2.5) oleyl ether carboxylic acid at nominal concentrations of 0 (control), 0.32, 1.0, 3.2, 10 and 32 mg/L under static conditions.

The measured concentrations were at the level of 95-234% of the initial during the test. Based on these results, the Time Weight Average concentrations were calculated: TWA were resp. 0.30, 0.84, 1.7, 1.9, 9.2 and 25 mg/L

The EC50 for growth rate inhibition (72h-ErC50) is 5.2 mg/L with a 95% confidence interval ranging from 4.1 to 6.5 mg/L. The EC10 for growth rate is 0.96 mg/L with a 95% confidence interval ranging from 0.6 to 1.4 mg/L

The EC50 for yield inhibition (72h-EyC50) was 1.4 mg/L with a 95% confidence interval ranging from 1.2 to 1.5 mg/L.

The 72h-NOEC for yield inhibition was 0.30 mg/L. The NOEC for growth rate inhibition could not be determined.