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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
23 March 2006, Annexe 5 corrected: 28 July 2011
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: batch 070602
- Expiration date of the lot/batch:06 Jun 2019
- Purity test date:31 jul 2017


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient, dark, dry
- Solubility and stability of the test substance in the solvent/vehicle: soluble

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Final dilution of a dissolved solid, stock liquid or gel: 15.0, 4.69, 1.46, 0.458, 0.143 mg/L

FORM AS APPLIED IN THE TEST (if different from that of starting material) : liquid

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
no
Details on test solutions:
Based on the results of a GLP range-finding test with nominal test item concentrations of 100, 10.0, 1.00, 0.100 mg/L and control, the following nominal test item concentrations were tested in the main test (spaced by a factor of 3.2): 15.0, 4.69, 1.46, 0.458, 0.143 mg/L and control.
The necessary amount of test item for preparing the stock solution S1 was weighed on a weighing scoop and transferred to a volumetric flask. OECD test medium was added up to the bench mark and the solution was homogenised by shaking. Afterwards the solution was clear and transparent. Lower test solutions were prepared by dilution of the 15 mg/L solution with test medium. Algae were added to each solution individually targeting nominal cell densities of 0.5 × 104 cells per mL in each solution

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata Hindak
- Strain/clone: SAG 61.81
- Source: MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany

ACCLIMATION
- Acclimation period: 3-4 days
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

Study design

Test type:
static
Water media type:
other: synthetic growth medium (OECD medium)
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
22.3 – 23.4 °C
pH:
7.81 – 8.33
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks (100 mL) with cellulose stoppers
- Type (delete if not applicable): closed
- Fill volume: ~ 50 mL test solution
- Aeration: CO2 supplied by continuous agitation
- Type of flow-through (e.g. peristaltic or proportional diluter): peristaltic
- Renewal rate of test solution (frequency/flow rate): none
- Initial cells density: 0.5 x 10E4 cells/mL
- Control end cells density: 45.11 x 10E4 cells/mL

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes (sterile cultures)
- semi-continuous growing. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures are ordered regularly from our commercial supplier.
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


TEST MEDIUM
- Source:OECD medium (according to Annex 3 of OECD 201)


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- pH control: 7.81 – 8.33
- Photoperiod: continuous
- Light intensity and quality: 75.9 µEm-2s-1 (mean)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence measurement (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)


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

- Range finding study: yes (non-GLP) with nominal test item concentrations of 100, 10.0, 1.00, 0.100 mg/L and control
- Test concentrations: 15.0, 4.69, 1.46, 0.458, 0.143 mg/L and control
- Results used to determine the conditions for the definitive study:After 72 h at termination of the test a dose response relation was observed for the inhibition of growth rate and yield from nominal test item concentrations of 0.100 to 100 mg/L. The inhibition of growth rate peaked at >100 % and the inhibition of yield peaked at 101.1 % at a nominal test item concentration of 100 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate is tested as the toxic reference item in a separate study twice a year to confirm the sensitivity of the test organism against compounds with known effects under the test conditions.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.66 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidential limits 1.86 – 3.42 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
4.44 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidential limits 3.45 – 5.35 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.62 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidential limits 8.32 – 11.1
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
4.69 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.46 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): none observed
- Other: No cells were observed at 15.0 mg/L nominal test item concentration
- Any stimulation of growth found in any treatment: yes, between 0 and 24 h of exposure from test solution of 0.143 to 4.69 mg/l and after 72h for test solutions of 0.143 and 0.458 mg/L.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 0.971 mg/L (growth rate)
- Other: NOEC: 0.320 mg/L
LOEC: 0.800 mg/L
Reported statistics and error estimates:
The statistical evaluation for the 72 hours period was performed for growth rate 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 NOEC and LOEC were determined by using a multiple comparison method (Dunnetts-t-test, left sided, for growth rate and yield). The EC10, 20, 50-values for growth rate were determined by probit analysis following the Gompertz. Due to statistical reasons inhibition-values below 0 % were set to zero. Only concentrations within a clear concentration response relation were used for calculations.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
X330 was tested following the OECD 201 guideline (2006, corrected 2011) without any deviation. The objective of this study was to determine the effects of the test Item on the growth of the single cell green alga Pseudokirchneriella subcapitata, to determine the no observed effect concentration (NOEC), to determine the lowest observed effect concentration (LOEC) and to determine the effect concentration (EC10, 20, 50), where possible.
Significant inhibitory effects were determined for growth rate at test item concentrations of 4.69 mg/L (nominal) and above. The LOEC was therefore determined to be 4.69 mg/L (nominal), the corresponding NOEC was set at 1.46 mg/L (nominal).
The EC10-value for growth rate (ErC10) was determined to be 2.66 mg/L (nominal). The EC20-value for growth rate (ErC20) was determined to be 4.44 mg/L (nominal).
The EC50-value for growth rate (ErC50) was determined to be 9.62 mg/L (nominal).
Executive summary:

The objective of this study was to determine the effects of X330 on the growth of the single cell green alga Pseudokirchneriella subcapitata, to determine the no observed effect concentration (NOEC), to determine the lowest observed effect concentration (LOEC) and to determine the effect concentration (EC10, 20, 50), where possible. Initial target cell densities of 0.5 × 104 cells/mL were employed for the individual replicates. The increase of cell numbers was assessed over a test period of 72 hours. Following a static range-finding test with nominal test item concentrations of 100, 10.0, 1.00, 0.100 mg/L and control a static main test with nominal test item concentrations of 15.0, 4.69, 1.46, 0.458, 0.143 mg/L and control was performed.

Six replicates were employed for the control and three for each test item concentration. The test was performed in 100 mL Erlenmeyer flasks each containing ~ 50 mL test solution. The pH was recorded at test start and test end. Temperature was measured continuously over the whole test period and recorded daily. Light intensity of the continuous illumination was measured at test start.

Analytical samples were taken and analysed in the main test from control and all test item concentrations at 0 hours (initial value) from fresh test solutions and after 72 hours from aged test solutions. Where possible NOEC and LOEC were determined by using a multiple comparison method and EC10, 20, 50-values were determined by probit analysis. All validity criteria were checked.

Toxicological endpoints for the test item:

   Test item (mg/L)
  nominal 
 ErC10 (Growth Rate) (1)  2.66
 ErC20 (1) 4.44 
 ErC50 (1) 9.62 
 NOEC (2) 1.46 
 LOEC (2) 4.69 

     

(1) Probit analysis following Gompertz distribution

(2) Following Dunnetts-t-test (left-sided, p<0.05) for growth rate and for yield