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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
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)
Version / remarks:
23 March 2006, Annex 5 corrected: 28 July 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: A static test with nominal loading rates of 100, 50.0, 25.0, 12.5, 6.25 mg/L
and control was performed.Analytical samples were taken and analysed from control and all test item loading rates at 0 hours (initial value) from fresh test solutions and after 72 hours from aged solutions.
- Sample storage conditions before analysis:After sampling, the test medium samples (50 mL) were stored deep-frozen (<= - 18 °C) until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
The test solutions were prepared in duplicate, firstly for pre-equilibration of the test vessels (see 4.5) without algae, secondly for the test itself with nominal algae cell densities of 0.5 × 104 cells per mL, whereby the algae were only added after stirring, settling and withdrawing. A stock solution (S1) was prepared by directly weighing 100 mg in 1000 mL test medium. This stock solution was stirred at 100 rpm in the dark at room temperature for 48 h (based on OECD Series on Testing and Assessment No. 23). After stirring an oily film was observed at the surface of the solution. Subsequently the undissolved test item was allowed to sediment and/or float for a period until the phases had separated. After the settling the necessary volume for the test was withdrawn via a Teflon tube from the medium level of the stock solution. The test item loading rates V1 – V4 were made by diluting the appropriate solutions with test medium to give the required test loading rates. Approximately 500 mL of the prepared solutions were transferred to each test vessel.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata Hindák (Sphaeropleales: Selenastraceae)
- Strain: SAG 61.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany
- Age of inoculum (at test initiation): 3 to 4 days before start of the test, test medium was inoculated with the test organism and held under test conditions in order to produce a pre-culture in the state of exponential growth
- Method of cultivation: test conditions

ACCLIMATION
- Acclimation period:3 to 4 days
- Culturing media and conditions (same as test or not): same
Culture conditions are as follows:
- Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 – 120 μEm-2s-1)
- Temperature: 21 - 24 °C
- Culture flasks: 100 mL Erlenmeyer flasks
- CO2 supply by shaking on a rotating shaker, approximately 105 rpm
Cells from this semi-continuous liquid stock culture were used for the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.5 – 23.0 °C
pH:
7.30 – 8.43
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks (100 mL) with aluminium caps were filled up with ~ 50 mL test solution.
- Initial cells density: 0.5 x 10^4 cells/mL
- Control end cells density: 43.99 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:AAP-Medium (according to Annex 3 of OECD 201)
- Culture medium different from test medium: no
- Intervals of water quality measurement: Measurements of pH-value were performed at t = 0 h and t = 72 h, the temperature was measured continuously and recorded at hours 0, 24, 48 and 72.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: Continuously from the side, 88.8 μEm-2s-1 (mean)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Fluorescence measurements were performed with a fluorescence microplate reader (infinite 200Pro) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 1.11.1.0).
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.007 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.007 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The morphology of the algae cells was observed microscopically at test end. The cells were considered normal for the control and up to and including a nominal test item loading rate of 100 mg/L.
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: not reported
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50: 1.64 mg/L
Reported statistics and error estimates:
The statistical evaluation for the 72 hours period was performed for growth rate and yield using SAS® (2002–2010). A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was evaluated by calculating the Levene Test. The NOELR and LOELR as well as NOEC and LOEC were determined by using a multiple comparison method (Dunnetts-t-test, left sided, for growth rate, Bonferroni-Holms corrected Welch test, left sided, for yield). The calculation of the ErL10, 20, 50/ ErC10, 20, 50 was not indicated since the inhibition was below 10 % at all test item concentrations for growth rate. The calculation of the EyL20, 50/ EyC20, 50 was not indicated since the inhibition was below 20 % at all test item concentrations for yield. The calculation of the EyL10/EyC10 was not indicated due to a missing concentration response relation and hence the database was inappropriate for probit analysis.

No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item loading rates, including the highest test item loading rate of 100 mg/L (nominal) at test end. Thus the overall LOELR/LOEC were not determinable, the overall NOELR was observed to be at 100 mg/L (nominal) and the corresponding overall NOEC based on actual concentrations was 0.00735 mg/L.

The EL10-, EL20- and EL50-value for growth rate and the EL20- and EL50-value for yield were considered to be > 100 mg/L (nominal). Due to an inhibition of yield below 20 % and a missing concentration response relation no reliable values were calculable and the EL10-value for yield was not determined. Based on actual concentrations the corresponding EC10-, EC20- and EC50-value for growth rate and the EC20- and EC50-value for yield were considered to be > 0.00735 mg/L and the EC10-value for yield was not determined.

Validity criteria fulfilled:
yes
Conclusions:
72h-ErL50: > 100 mg/L
72h-ErC50: > 100 mg/L
Executive summary:

In the Klimisch 1 GLP study from Obert-Rauser (2017) the acute toxicity of 2,6-Di-tert-butyl-4-nonylphenol on the Single Cell Green Alga Pseudokirchneriella subcapitata Hindák was determined in an 78 hour static test according to OECD 201. The test was performed with nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg test item/L prepared as WAF and a blank control. Six control replicates and 3 replicates for each treated test group were set up. Cell density was determined every 24 hours by fluorescence measurements. After 72 hours 0, -3.1, 0.2, 4.3, -0.2 and -4.7% inhibition of the growth rate relative to the control was observed at nominal loading rates of 6.25, 12.5, 25, 50 and 100 mg test item /L, respectively. No statistical significant differences were found between the treated test groups and the control. Analytical measurements resulted in following geometric mean measured concentrations for the 6.25, 12.5, 25, 50 and 100 mg test item/L treatment levels: 0.00143, 0.00156, 0.00265, 0.00515 and 0.00735 mg/L, respectively. Since no adverse effects were observed the data were based on loading rates. The ErL10 and ErL50 were > 100 mg/L.

The results are considered relevant and reliable for the risk assessment

Description of key information

72h-ErL50: > 100 mg/L

72h-ErC50: > 100 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

In the Klimisch 1 GLP study from Obert-Rauser (2017) the acute toxicity of 2,6-Di-tert-butyl-4-nonylphenol on the Single Cell Green Alga Pseudokirchneriella subcapitata Hindák was determined in an 78 hour static test according to OECD 201. The test was performed with nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg test item/L prepared as WAF and a blank control. Six control replicates and 3 replicates for each treated test group were set up. Cell density was determined every 24 hours by fluorescence measurements. After 72 hours 0, -3.1, 0.2, 4.3, -0.2 and -4.7% inhibition of the growth rate relative to the control was observed at nominal loading rates of 6.25, 12.5, 25, 50 and 100 mg test item /L, respectively. No statistical significant differences were found between the treated test groups and the control. Analytical measurements resulted in following geometric mean measured concentrations for the 6.25, 12.5, 25, 50 and 100 mg test item/L treatment levels: 0.00143, 0.00156, 0.00265, 0.00515 and 0.00735 mg/L, respectively. Since no adverse effects were observed the data were based on loading rates. The ErL10 and ErL50 were >100 mg/L, i.e., the test item is predicted to have no toxic effects at saturation.

The results are considered relevant and reliable for the risk assessment