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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Test started with algae in stationary phase.
Test performed for 14 days instead 72 hours.
Counting of algae every second day (not daily) in Bürker´s chamber.
Light intensity 2000 lx.
Spacing between concentrations factor ten (which is considered to be a minor deviation).
Effect (LC50) based on dry weight at the end of the test and not on growth rate.
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Chlorococcum sp.
Details on test organisms:
TEST ORGANISM
- Strain: No. 564
- Source (laboratory, culture collection): Institute of Zootechnics at Zator, Poland
- Age of inoculum (at test initiation): not provided, but typically a lag-phase was observed in the experiments indicating that the culture was not exponentially growing or that the cultuer conditions have changed.
- Method of cultivation: not provided, but typically a lag-phase was observed in the experiments indicating that the culture was not exponentially growing or that the cultuer conditions have changed.

ACCLIMATION
- Acclimation period: not provided
- Culturing media and conditions (same as test or not): not provided, but typically a lag-phase was observed in the experiments indicating that the culture was not exponentially growing or that the cultuer conditions have changed.
- Any deformed or abnormal cells observed: not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
14 d
Test temperature:
23-25°C
Nominal and measured concentrations:
control, 0.01, 0.1, 1, 10 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL glass flasks
- Material, size, headspace, fill volume: 150 mL
- Initial cells density: 0.01 - 0.02 g/L, about 0.5*10^6 cells/mL as determined from Fig. 11
- Control end cells density: about 21.8*10^6 cells/mL as determined from Fig. 11
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates):not reported

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: L5m medium (Jankowski 1964)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: L5m medium (Jankowski 1964)
- Culture medium different from test medium: no
- Intervals of water quality measurement: measurements not reported

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: no
- Photoperiod: assumed to be continuous illumination
- Light intensity and quality: 2000 lx fluorescence lamps of the day-light type
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting with Bürker´s chamber every two days; determination of the dry weight at the end of the test
- Chlorophyll measurement: no
- Other: assessment of the appearence of the cells at the end of the test

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: not applicable
Reference substance (positive control):
no
Key result
Duration:
14 d
Dose descriptor:
IC50
Effect conc.:
6.5 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): based on Fig. 11: in teh first days: yes, thereafter: no
- Observation of abnormalities (for algal test):
- Unusual cell shape: Lethal doses caused swollen cells with destruced chloroplasts devoid of green color, later the cells were described as being "empty"
- Colour differences: Lethal doses caused discoloration while the medium remained clear
- Any stimulation of growth found in any treatment: no
- Effect concentrations exceeding solubility of substance in test medium: no
Validity criteria fulfilled:
not applicable
Remarks:
Test performed before guidance documents were established, but re-evaluation of the data confirmed that the validity critera of the OECD 201 (2011) which could be checked with the data available were met.
Conclusions:
Chlorococcum sp. 14-d EC50: 6.5 µg/L
Executive summary:

In the study from Bednarz (1981) the toxicity of monuron on the unicellular green algae Chlorococcum sp. was determined in a 14 day test. The test was performed as with five monuron concentrations, e.g, 0.1, 1, 10, 100 and 1000 µg/L and a control group. The test was performed in 300 mL flasks filled with 150 mL test solution. The light intensity was 2000 lx provided by fluorescence lamps of the day-light type. The cultures were put on a shaker. Cell density was determined every second day. The EC50 was determined based on the biomass determined after 14 days exposure. The EC50 based on biomass was determined to be 6.5 µg/L.

 

In order to the check the relevance of the data and the obtained EC50, the cell numbers were read -out from a figure which provided the growth curves of the test groups. A typical sigmoid growth curve was observed. A lag-phase could not be excluded for the time interval from day 0 to day 1. Therefore, this time interval was not considered in the further evaluation. From day 1 to day 6, the growth of the control culture was exponential. Thereafter, the increase of cell density slowed down. The evaluation of the growth rate was based on the exponential phase from day 1 to day 6. For that time interval the day to day growth rates were calculated for all test groups.

The daily control growth rates from day 1 to day 6 were used to check if the data fulfil the validity criteria of the most recent OECD 201 (2011). In that time interval, the cell density increased by a factor of 16 and hence fulfilled the first validity criterion which requests an 16-fold increase of cell number of the control group during the analysed time interval. The C.V. of the section-by-section growth rate of the control was 15% and fulfilled the validity criterion of being <35% for this second validity criterion. Since no information on replicates is available, the third validity criterion (C.V. of average specific growth rates for the replicates) was not applicable. The analysis of the validity criteria indicates that the results from this test are reliable with restrictions.

The obtained ErC10 and ErC50 values were 1.9 and 8.0 µg/L. These data are almost identical to the EyC50 based on biomass after 14 days of 6.5 µg/L as reported by Bednarz (1981).

It can be concluded that the re-evaluation of the Chlorococcum sp. data as provided by Bednarz (1981) demonstrated that

·        the data are reliable with restrictions and that

·        the obtained endpoints are robust.

Therefore, the obtained results are considered usable for the risk assessment. The risk assessment will be based on the EC50 of 6.5 µg/L.

Description of key information

most sensitive EC50-value: 6.5 µg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
6.5 µg/L

Additional information

 

Bednarz (1981) analysed the toxicity of monuron on in total 12 algal/cyanobacteria species (9 green algae and 3 cyanobacteria species). Of all the species tested the unicellular green algae Chlorococcum sp. was the most sensitive one having an EC50 of 6.5 µg/L (based on biomass after 14 days). Due to its sensitivity, this species is of special interest for the risk assessment of monuron. Therefore, the results for this species were assessed in detail for relevance and reliability.

The tests from Bednarz were performed from 1974 to 1979, i.e., before the modern guidelines like the OECD 201 were established. Hence, the test design and the evaluation of the data as performed by Bednarz deviates in some aspects from the guidance given in the new guidelines. As major deviations the test was performed for 14 days instead 72 hours and the evaluation was based on biomass at the end of the test and not the growth rate. In order to evaluate the impact of the 14-day exposure period and the use of the biomass instead of the growth rate on the evaluation of the study, the growth curves for Chlorococcum sp. (cells/mL vs. time) as provided in Fig. 11 in Bednarz (1981) were re-analysed in detail.

That re-analysis demonstrated that the results are reliable with restriction since two of three validity criteria listed in the OECD 201 (2011) were met. The third criterion i.e., the CV of the average specific growth rates could not be calculatedsinceno control replicate data were provided.

It can be concluded that the re-evaluation of the Chlorococcum sp. data as provided by Bednarz (1981) demonstrated that

·        the data are reliable with restrictions and that

·        the obtained endpoints are robust.

Therefore, the obtained results are considered usable for the risk assessment. The risk assessment will be based on the EC50 of 6.5 µg/L.