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Ecotoxicological information

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:
2013-11-25 - 2014-01-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Identification: Dimethylpiperazine
- Description: Pale yellow liquid
- Batch: 1903-3/2012
- Purity: 99.82%
- Expiry date: 31 December 2014
- Storage conditions: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis.
- Sampling method: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.
Vehicle:
no
Details on test solutions:
- 100 mg/L stock solution (50 mg in 500 mL culture medium)
- Lower test concentrations prepared by serial dilution using the primary stock solution
- The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute,
Oban, Argyll, Scotland
- Age of inoculum (at test initiation): not stated

ACCLIMATION
- Acclimation period: none
- Culturing media and conditions (same as test or not): The culture medium used for both the range-finding and definitive tests was the same as that
used to maintain the stock culture
- Any deformed or abnormal cells observed: not noted
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none
Hardness:
not reported
Test temperature:
24 +/- 1 °C
pH:
Definitive test:
Start: 7.5
End: 7.8

It was noted during the range-finding test that the pH of the test preparations at 0 hours increased from pH 7.4 at 0.10 mg/L through to pH 9.3 at 100 mg/L. After discussion with the Sponsor, and following the recommendations of the test guideline, it was considered appropriate to adjust the pH of the definitive test preparations prior to the addition of algal cells at 0 hours to pH 7.5 in order to overcome any possible effects of the alkaline test conditions.
Dissolved oxygen:
not reported
Salinity:
not applicable
Nominal and measured concentrations:
Range finder:
0.10, 1.0, 10 and 100 mg/L
Definitive:
Nominal concentrations: control, 1.0, 3.2, 10, 32, 100 mg/L
Determined concentrations:
at 0 hrs: < LOQ, 1.0, 3.23, 9.77, 31.3, 99.7 mg test item/L
at 72 hrs: < LOQ, 0.993, 8.4*, 10.0, 30.7, 100 mg test item/L


* original sample gave a higher than expected result outside the calibration range, therefore the frozen duplicate sample was analyzed.
% of nominal concentration range was 96 - 101% except for the higher value of 8.4 mg/L (% of nominal concentration was 263).
Details on test conditions:
TEST SYSTEM
- Test vessel: glass conical flask
- Test vessels covered or not: closed with polyurethane foam bungs and incubated (INFORS Multitron, Version 2 incubator)
- Material, size, headspace, fill volume: glass, 250 mL, 100 mL fill volume
- Aeration: only by nominal orbital shaking at 100-150 rpm
- Initial cells density: 5x 10^3
- Control end cells density: 10^4-10^5 cells/mL. Cell concentration of the control cultures increased by a factor of 145 after 72 hours
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- One blank vessel (without algal inoculum) was incubated for each test concentration and the culture medium control (for background corrections)
- Random position in incubator, changed daily

GROWTH MEDIUM
- Standard medium used: yes (AAP medium)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: with 1M HCl to appropriate range
- Photoperiod: 24 h light
- Light intensity and quality: warm white lighting (380 – 730 nm), 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): algal cell density (calculated parameters: biomass and growth rate) was determined after 24, 48 and 72 h
- Determination of cell concentrations: Coulter electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.1-3.3
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
NOEC
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:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: not determined
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none observed
- Effect concentrations exceeding solubility of substance in test medium: none
Biomass based effect concentrations:
- EbC10 = 0.57 mg/L (95% CI = 0.36-1.7 mg/L)
- EbC20 = 1.0 mg/L (95% CI = 0.6-1.8 mg/L)
- EbC50 = 2.5 mg/L (95% CI = 2.0-2.9 mg/L)
- NOEC = 0.36 mg/L
- LOEC = 1.2 mg/L
Remaining growth rate based effect concentrations:
- EbC20 = 2.8 mg/L (95% CI = 2.5-3.2 mg/L)
- LOEC = 3.7 mg/L
Reported statistics and error estimates:
NOEC and LOEC values were determined by one-way analysis of variance and Dunnett's procedure to identify significant differences (p<0.05) between individual treatments and the culture medium control.
EC50, EC20 and EC10 values and their associated confidence intervals were calculated using the Linear Interpolation method.
Validity criteria fulfilled:
yes
Conclusions:
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal with the exception of the 3.2 mg/L test sample at 72 hours. Initial analysis showed the response to be outside of the linear range whilst analysis of the duplicate sample showed a measured concentration of 8.4 mg/L was obtained. Given that a measured concentration of 101% of nominal was observed at 0 hours it was considered that the test solution had been correctly prepared and that possible post study contamination had occurred. As such, given that the test concentration was below the No Observed Effect Concentration it was considered appropriate to base the results on the nominal test concentrations only. Exposure of Pseudokirchneriella subcapitata to the test item gave a 72-hr EC50 value > 100 mg/L. The 72-hr NOEC was 100 mg/L.

Description of key information

A full study was performed under GLP conditions and according to OECD TG 201 (Huntsman, 2014). The 72-h EC50 for the test substance based on growth rate was > 100 mg/L. The NOEC was 100 mg/L. As a worst-case, a value of 72 -h EC50 = 100 mg/L was used for the PNEC derivation.

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 a 72-hour static test with the green alga Pseudokirchnerella subcapitata according to the OED 201 guideline, the toxicity of the test substance to algae was investigated (Vrijenhoef, 2014). Tested concentrations were: 3.2, 10, 32, 100 mg/L. Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal with the exception of the 3.2 mg/L test sample at 72 hours. Given that a measured concentration of 101% of the nominal concentration was observed at 0 hours it was considered that the test solution had been correctly prepared. As such, given that the test concentration was far below the NOEC it was considered appropriate to base the results on the nominal test concentrations only.

After 72 hours an EC50 of > 100 mg/L for the test substance was estimated for the growth rate, related to the nominal concentration. The 72-h NOEC was 100 mg/L.