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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:
2001-01-08 to 2001-06-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, 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:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ATE-660, 0011-001 MXL

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: see manufacturer safety data sheet.
- Stability under test conditions: soluble, stable substances

OTHER SPECIFICS:
- Chemical name: CUMYL PEROXYNEODECANOATE 70 % - ISODODECANE 30 %
- CAS number: 26748-47-0 and 31807-55-3
- appearance : colourless liquid
- purity : 71.5 % peroxyde
- water solubility : 0.2 mg/L
Analytical monitoring:
yes
Details on sampling:
At the beginning of the definitive test and prior to addition the organisms, test solutions are sampled and chemically analysed in order to verify that initial concentrations are equivalent to nominal concentrations (soluble, stable substances).
- Concentrations: 9.9; 17.3; 30.9; 55.9 and 100 mg/L
- Sampling method: The test solutions were prepared according to WAF principle in order to obtain the test solutions at concentrations varying from 9.9 mg/L to 100 mg/L. After 24h, 48h, and 72h of incubation, about 1 mL was sampled from each test flask under agitation and introduced into culture tubes. After 24 and 48 hours test flasks were replaced in the same position in the rotary shaker.
- Dissolved O2 and pH were measured in the control and the highest concentration, in non inoculated flask at the beginning of the test and in an inoculated flask at the end of the test.
- Sample storage conditions before analysis: cold and dark
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test solutions were prepared according to WAF principle in order to obtain the test solutions at concentrations varying from 9.9 mg/L to 100 mg/L. Individual solutions were prepared by vigorously mixing 9.9; 17.3; 30.9; 55.9 and 100 mg of cumylperoxyneodecanoate in isododecane with 1 litre of dilution water. After 66 hours of stirring, excess of non dissolved test item was removed by filtration on Millipore membranes (HV 0.45 μm). Thus the prepared solutions correspond to WAF of respectively mixing 9.9; 17.3; 30.9; 55.9 and 100 mg/L of cumylperoxyneodecanoate in isododecane.
Since the test was performed using hermetically closed flasks, NaHC03 0.3 g/l and HEPES buffer 6 mM (4-(2-Hydroxyethyl)-l piperazineethanesulfonic acid, sodium salt) were added to dilution wateras described in ISO/FDIS 14442: Water Quality - Guidance for algal growth inhibition tests with poorly soluble organic and inorganic solids, volatile compounds, heavy metals and waste water.
- Differential loading: 9.9; 17.3; 30.9; 55.9 and 100 mg/L
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): excess of non dissolved test item was removed by filtration on Millipore membranes (HV 0.45 μm).
The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear and colourless over the period of the test. No precipitation was observed at the end of the test.

For the preliminary test, a stock solution of the test item diluted in water was prepared 22.5 hours before the beginning of the test (time 0) by mixing 50.4 mg of cumylperoxyneodecanoate in isododecane in 0.5 litre of dilution water. Volumetric flasks were filled with the required volumes of the test item stock solution and dilution water in order to obtain the test solutions at concentrations varying from 0.1 mg/L to 100 mg/L. Flasks were stoppered with PTFE bungs and sealed with aluminium caps, and placed in the phytoculture cupboard.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): obtained from the Culture Centre of Algae and Protozoa (Ambleside, UK)
- Method of cultivation: Two flasks, each containing approximately 100 mL of axenic stock culture of algae are incubated at 23 ± 1 °C under lighting (photoperiod: 16 hours of illumination, 8 hours of darkness), and continuous aeration (air filtered at 0.22 μm).
These stock cultures are renewed every week, using two new cultures.

ACCLIMATION
- Acclimation period: Four days before the beginning of the study two pre-cultures were prepared by inoculating each stock suspension of algae (5 mL) into sterile dilution water (500 mL). The pre-cultures were incubated under the same conditions as those used for the stock cultures. Only one of the two pre-cultures was used to inoculate the test flasks; the second one was to be used only if the first one was damaged. At the beginning of the test, the cell concentration of the pre-culture was determined. The
result was used to calculate the volume needed and introduced into each test flask in order to get an initial cell concentration equal to 1x10^4 cells/mL.
- The cell density of the pre-culture was about 3.03 x 10^6 cells/mL for the preliminary test and about 7.29 x 10^6 cells/mL for the definitive test.
- Culturing media and conditions (same as test or not): Same, but since the test was performed using hermetically closed flasks, NaHC03 0.3 g/L and HEPES buffer 6 mM (4-(2-Hydroxyethyl)-l piperazineethanesulfonic acid, sodium salt) were added to dilution water (see annex 1) as described in ISO/FDIS 14442: Water Quality - Guidance for algal growth inhibition tests with poorly soluble organic and inorganic solids, volatile compounds, heavy metals and waste water.
- Any deformed or abnormal cells observed:
- The quality of the stock culture was verified for the absence of microorganisms and deformed cells under microscopic observation before use.
- the normal form of the unicellular algae is a crescent shaped cell with an average length of 5-10 μm.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24.1 ± 0.5 °C
pH:
7.18 - 7.75 (Measurements were carried out in non inoculated solutions at T0 and in inoculated solutions after 72 hours of incubation)
Dissolved oxygen:
8.3 - 9.8 mg/L (Measurements were carried out in non inoculated solutions at T0 and in inoculated solutions after 72 hours of incubation)
Nominal and measured concentrations:
Loading rate (WAF), nominal: 9.9, 17.3, 30.9, 55.9, 100 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass bottles stoppered with PTFE bungs and sealed with aluminium caps.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 120 mL
- Aeration: yes
- Initial cells density: initial cell concentration was equal to 1 x 10^4 cells/mL
- Control end cells density: yes
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3


GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Water: ultra-pure water was used for the preparation of dilution water (resistivity > 18 MΩ).
- Since the test was performed using hermetically closed flasks, NaHC03 0.3 g/l and HEPES buffer 6 mM (4-(2-Hydroxyethyl)-l piperazineethanesulfonic acid, sodium salt) were added to dilution water as described in ISO/FDIS 14442
- Dilution water is prepared according to the protocol described in paragraph 1.6.1.of the method C.3.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes
- Photoperiod: 16 hours of illumination, 8 hours of darkness


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of algae concentration was carried out using an optical microscope equipped with an haemacytometer (Malassez cell counter) for the definitive test, and by cytofluorimetry with a Cytofluor 2350 for the preliminary test.
- Chlorophyll measurement: After 24h, 48h, and 72h of incubation, a volume of 200 μl was sampled from each test flask, pipetted into a quartz microplaque. Fluorescence was then determined with a cytofluorimeter, and by comparison with a calibration range, cell density was determined, according to the measured fluorescence.


TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 0.1 to 100 mg/L
- Results used to determine the conditions for the definitive study: yes
The preliminary test was performed in a phytoculture cabinet that allows test flasks to be incubate under precise conditions: temperature is set to 23 °C; flasks were continuously shaken with a rotation of 20 rpm , and constantly illuminated by 8 fluorescent tubes. The temperature, measured continuously in one test flask, was maintained between 21 and 25 °C.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (geom. mean)
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): Microscopic observation confirmed that the algae appeared normal at the end of the test in comparison to the control.
- Unusual cell shape: No
- Cell density: the increase in cell density (R), measured in the control solution between the end and the beginning of the test, was greater than a factor of 16 (R = 188).
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50: 1.90 mg/L (obtained on January 1, 2001)
- EbC50: 0.86 mg/L (obtained on January 1, 2001)
Reported statistics and error estimates:
The No Observed Effect Loading (NOEL) was determined using a statistical analytical method (ANOVA and Dunnett test).

Table 2: Effective concentrations and NOEL.

       Effective loading and NOEL (mg/L)
   Value

 95 % Cl

       Cell growth
 EbL50 - 72h  >100  ND
 EbL10 - 72h   >100   ND
 NOELb   >100   ND
       Growth rate
  ErL50- 72h   >100   ND
  ErL10- 72h   >100   ND
  NOELr   >100   ND

Cl: 95 % confident interval; ND: not determined

Validity criteria fulfilled:
yes
Conclusions:
For Pseudokirchneriella subcapitata, an ErL50 value (based on growth rate) of > 100 mg/L was determined. The NOEL was found to be > 100 mg/L.
Executive summary:

In a 72–hr acute toxicity study, the cultures of freshwater algae Pseudokirchneriella subcapitata were exposed to cumylperoxyneodecanoate (71.5% in isododecane) at nominal concentrations of 9.9, 17.3, 30.9, 55.9, 100 mg/L under static conditions in accordance with the method C.3 described in Directive 92/69/EEC of the European Commission and in the guideline 201 of the OECD. 

The test item is a mixture of 2 constituents with different solubilities and physico­chemical properties. Therefore, test solutions were prepared according to the WAF methodology (water accommodated fraction) described by OECD.

Concentrations of the test item were measured by HPLC coupled with mass spectrometry. Cumylperoxyneodecanoate was not found in test solutions at time 0 and 72 h. Therefore, the degradation product neodecanoic acid was analysed. The relative concentration of neodecanoic acid was found to be constant during the test period. Since no commercial standard of neodecanoic acid was available, the exact quantification of this sub-product was not carried out. The results are expressed as equivalents of an internal standard (lauric acid). They are used to correlate the percentage of final over initial concentrations. The concentrations were maintained within the designated Iimit of 80 % of the initial concentrations in the test solution. Thus, effective concentrations have been calculated using the nominal loading of cumyl peroxyneodecanoate in isododecane. The % growth inhibition in the treated algal culture as compared to the control ranged from -4.02 % at a concentration of 9.9 mg/L to 1.15 % at a concentration of 100 mg/L.

There were no compound related phytotoxic effects.  

This toxicity study is classified as acceptable and satisfies the guideline requirements for an algae growth inhibition toxicity study.  

Results Synopsis

 

Test Organism: Raphidocelis subcapitata

Test Type (Flowthrough, Static, Static Renewal): static

Specific Growth Rate:

72 -hr ErL50  >100                

72 -hr ErL10  >100  

72 -hr NOEL >100      

         

Endpoint(s) Effected: Specific growth rate, yield

Description of key information

For Pseudokirchneriella subcapitata, an ErL50 value (based on growth rate) of > 100 mg/L was determined. The NOEL was found to be > 100 mg/L (EU method C.3, OECD 201).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information