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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Analytical monitoring:
Details on sampling:
All treatments were analytically monitored at the beginning and the end of the test.
Details on test solutions:
Tests solutions were prepared from dilutions of a stock solution. A known volume of test item (50.0 mg) was poured into a flask, the volume was then made up to 500 mL with test medium. The solution was kept under high speed stirring at ambient temperature during approximately 22 hours with a magnetic stir bar. No filtration was necessary before dilution.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test organism used for the study was Pseudokirchneriella subcapitata, Strain No. CCAP 278/4, supplied by the Culture Centre of Algae and Protozoa (Ambleside, UK). Transfers of P. subcapitata were made regularly to provide suitable subcultures. The algae were cultivated under standardized conditions as described in Annex 2 of the OECD 201 guideline. The quality of the stock culture was checked for the absence of micro-organisms and deformed or abnormal cells under microscopic observation before use.
Three days before the start of the exposure, two pre-cultures were prepared by inoculating each stock suspension of algae (1 mL) into sterile dilution water (100 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 for the study. The second one was only used if the first one was damaged.
At the beginning of the test, the cell density of the pre-culture was determined. The result was used to calculate the volume to be introduced into each test flask in order to obtain an initial cell concentration of 5000 cells/mL as recommended in the OECD 201 guideline.
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Remarks on exposure duration:
Post exposure observation period:
Not measured.
Test temperature:
22.7°C - 23.7°C (min-max)
7.8 - 8.0 (min-max)
Dissolved oxygen:
8.6 - 9.3 (min-max)
Not measured.
Not measured.
Nominal and measured concentrations:
Nominal concentrations: 0 ; 0.43 ; 0.94 ; 2.07 ; 4.54 ; 10.00 mg/L
Measured concentrations at 0h: 0 ; 0.43 ; 0.97 ; 2.04 ; 4.49 ; 9.87 mg/L
Measured concentrations at 72h: 0 ; 0.41 ; 0.90 ; 1.98 ; 4.37 ; 9.63 mg/L
Since almost no concentrations deviation occurred, results were expressed according to nominal concentrations.
Details on test conditions:
The test was conducted at the following nominal test item concentrations: 0.43, 0.94, 2.07, 4.54 and 10.00 mg/L. The range of concentrations was obtained from dilutions of the stock solution. Three vessels were prepared at each test concentration and six vessels for the control group; each vessel was inoculated with 5000 cells/mL.
After 24 and 48h of incubation, aliquots of 200 µL were sampled from each inoculated test flask and pipetted into a microplate. After 72 h, the samples with high cell density were diluted to ¼ (50 µL + 150 µL of filtered dilution water) and pipetted into a microplate. Microplates were then analysed using a flow cytometer (Guava easyCyte™ flow cytometer Merck Millipore). Time between sampling and measurement was approximately 15 – 30 min.
Algal cell concentrations were measured in each flask at 24, 48 and 72h using flow cytometry (Guava easyCyte™ flow cytometer Merck Millipore). Cell concentrations were determined using 96 wells single use microplates and a laser beam at 488 nm. The cytometer is calibrated every week using the Guava check kit. Before each measurement, settings were adjusted. Non-algal particles were excluded from the analysis by setting an acquisition value threshold. This threshold was set up after analysis of cytograms with no threshold where a clear discrimination was observed between the algal population and the other events. Therefore, only the events with the same size than alga were used to assess the toxic effects.
The number of events counted was set up to 1500 to 3000 depending on the algal population. To minimize the number of coincident events, the analysed cell concentration was less than 500 cells/µL. Data processing was carried out using “Insight Software” (Guava easyCyte™ flow cytometer Merck Millipore).
The pH and dissolved oxygen concentrations were measured in all test solutions, in non-inoculated flask at the beginning of the test and in inoculated flasks at the end of the test. The temperature in the incubator was continuously recorded throughout the test.
Reference substance (positive control):
Key result
72 h
Dose descriptor:
Effect conc.:
6.9 mg/L
Nominal / measured:
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
72 h
Dose descriptor:
Effect conc.:
4 mg/L
Nominal / measured:
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
See table 1.
Results with reference substance (positive control):
Potassium dichromate : 72h-EC50 = 0.76 mg/L
Reported statistics and error estimates:
Statistical analysis was performed with the software ToxRat®. A Probit analysis using linear max. likelihood regression was statistically considered to be more adapted to the raw data obtained.

Table 1: mean growth rate of algae per treatment throughout the test. Figures are expressed as per 10 000 cells/mL.

Concentrations (mg/L)

Cells density 24h

Cell density 48 h

Cell density 72h

Growth rate (0-72h)

Growth rate inhibition (%)





































Validity criteria fulfilled:
This study was designed to determine the effects of 1,3 -dibutylthiourea to Pseudokirchneriella subcapitata over a 72h period under static conditions, according to the OECD 201 Guideline. EC50 was 6.9 mg/L and EC10 was 4.0 mg/L, both for growth rate inhibition.
Executive summary:

This study was designed to determine the effects of the test item on the growth of Pseudokirchneriella subcapitata in a 72-hour test according to the OECD 201 Guideline. Tests solutions were prepared from dilutions of a stock solution. Chemical analysis of the test item showed the stability of the substance over the 72-hour test period. Based on these results, the exposure concentrations were based on the nominal concentrations.

The results are reported as growth rate inhibition. The concentrations that result in a 10 and 50% reduction in growth rate (72h-ErC10 and 72h-ErC50, respectively) were determined. ErC50 was 6.9 mg/L and ErC10 was 4.0 mg/L.


The definitive test met the validity criteria of the guideline:

- The biomass in the control cultures increased exponentially by a factor of 79.6 within the 72-hour test period (required: at last 16-fold)

- The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures was max 26.3% (required: < 35%)

- The coefficient of variation of average specific growth rates during the whole test period in the control cultures was 4.0% (required: < 7%)

Description of key information

One OECD 201 study is available for acute toxicity of 1,3 -dibutylthiourea to algae. ErC50 was 6.9 mg/L and ErC10 was 4.0 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
6.9 mg/L
EC10 or NOEC for freshwater algae:
4 mg/L

Additional information