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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:
Algae were exposed to the test substance from 5 - 9 December 2006; The test solutions were analysed on 8 and 12 December.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
See "details on analytical methods"
Vehicle:
no
Details on test solutions:
Preparation of test solutions
On the day of test initiation a stock solution of 100.8 mg/I was prepared by dissolving 50.4 mg F1EC in 0.5 L algal medium. Based on a preliminary test with F1EC and algae the study was conducted at 0, 1.0, 2.2, 5.0, 11 and 25 mg/L. Test solutions were prepared on day 0 in three sterile 250-mL Erlenmeyer flasks per concentration. Six control Erlenmeyer flasks were prepared. The algae were exposed to the test solutions during 72 hours. All test solutions were clear and particles of the test substance were not visible.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Culture of the freshwater green alga Selenastrum capricornutum (strain ATCC 22662) was received on 19 January 2004 from the Culture Collection of Algae and Protozoa, Am.bleside, Cumbria, UK. Erlenmeyer flasks with medium were inoculated with the algae on 19 January 2004. At the Health, Environment & Safety Department of Solvay Pharmaceuticals the algae are cultured in 100-ml Erlenmeyer flasks with 50 ml filter sterilised medium. Each week a volume of 0.5 - 2 ml of this culture is transferred into fresh algal medium. The preculture used for this study was transferred into fresh algal medium 4 days prior to the start of the test. The culture chamber is illuminated 24 hours a day with fluorescent lamps.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
See details on results
pH:
The pH ranged between 7.5 and 8.5.
Dissolved oxygen:
No data
Salinity:
Not relevant
Conductivity:
No data
Nominal and measured concentrations:
The nominal concentrations were as follows (mean mesured concentrations in parenthesis):
1 (1.2), 2.2 (2.4), 5.0 (5.0), 11 (10.8) and 25 (25.3) mg/L.
Details on test conditions:
Test initiation and environmental conditions:
Before test initiation, the algal density in the preculture was determined by using a counting chamber (Neubauer improved) and a microscope (magnification 200 times). The cell density appeared to be 283 x 10E4 cells/mL. At test initiation a quantity of 353 μL of the preculture was added to all Erlenmeyer flasks. This resulted in an initial cell density of 1.0 x 10E4 ceIls/mL.
During the test period, the Erlenmeyer flasks were placed in a shaking incubator with a temperature set point of 24 ± 1°C. The shaking speed in the incubator was 90 - 110 rotations per minute. A randomised design was made by computer to determine the sequence of the test flasks in the shaking incubator. The Erlenmeyer flasks in the shaking incubator were illuminated continuously during the experiment with fluorescent lamps.

Biological observations and measurements:
To determine the effect of F1EC on growth of the algae, the absorption of the test solutions at 750 nm was measured during the test. The absorption was measured at 24, 48 and 72 hours after the start of the test in all Erlenmeyer flasks using a spectrophotometer (HP 8453) and a cuvette with a path length of 5 cm. The volume (10mL), used for measurement of the absorption, was not returned to the Erlenmeyer flasks.
A calibration line was used to determine the cell density of the test solutions based on measurements of the absorption (linear interpolation method). The algal preculture was used to prepare the calibration line.
To prepare calibration standards, volumes of 0.039; 0.078; 0.156; 0.313; 0.625; 1.25; 2.5; 5.0 and 10 mL of the preculture were made up to an end volume of 10.0 mL with algal medium. The calibration line was stored in the spectrophotometer.
On day 0 the light intensity at the photosynthetically effective wavelength range of 400 nm to 700 nm was measured in the shaking incubator at the level of the Erlenmeyer flasks. The light intensity at the level of the algae should be 60 - 120 μE/m2.s (ISO, 1989).
During the test the temperature of the thermostated incubator was recorded once every hour.
The pH of the algal medium and the highest test concentration was measured at test initiation. The pH of the test solutions was measured at test termination in one test vessel per concentration.

Calculations
The biomass integral was calculated and as it was based on the duration of the complete test, the growth rate was also based on days 0-3 (ISO, 1989). This calculation method was slightly different from OECD Guideline 201 (OECD, 1984).
The cell density measurements were used to calculate the biomass integral (day 3) and growth rate (days 0-3) for each Erlenmeyer flask. These results were used to determine the mean biomass integral and mean growth rate for each test concentration. Hereafter the inhibition (%) of the biomass integral and the inhibition (%) of the growth rate was calculated for each test concentration.
Linear interpolation, with concentration on a logarithmic scale (base = 10), was used to determine the 72hEC50 for biomass integral and growth rate inhibition. The No Observed Effect Concentration (NOEC), based on biomass integral, was determined with Williams' Test (Williams, 1972) using the statistical program SAS.

Validity criteria
The test is considered to be valid if the algal density in the control has increased at least 16 times after 3 days (OECD, 1984), and the pH in the control cultures shall not increase by more than 1.5 units.

Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
6.3 mg/L
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
32 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Statistically significant NOEC
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Biologically and statistically significant NOEC
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Biologically significant NOEC
Details on results:
Chemical analysis:
In the standards with a nominal F1EC concentration of 2.5 mg/L algal medium a mean concentration of 0.465 (± 0.01) mg fluoride/L was found (corrected for the blank) which corresponds to 2.58 mg/L F1EC. In the standards with a nominal F1EC concentration of 5.0 mg/L a mean concentration of 0.892 (± 0.02) mg/L fluoride was found which corresponds to 4.96 mg/L FIEC. The coefficient of variation of the standards at 2.5 and 5.0 mg/L was 3 .1 % and 2.2 %, respectively which meets the validity criterion of < 10%. The measured concentration of fluoride is in accordance with the validity criterion of 18 ± 5%. For this reason the test is suitable for the determination of F1EC by measuring the fluoride concentration and a conversion factor of 5.56 (100/18) was used to calculate the concentration F1EC.
The results of the analysis are summarised in Table 1.The difference between the nominal and the mean measured concentrations was < 20% and for this reason the endpoints of the test were based on nominal concentrations.

Cell density, biomass integral and irrowth rate:
The mean measured algal cell densities are given in Table 2. The data show that the cell density increased 121 - 155 times in the controls, which meets the validity criterion of a minimal 16 times increase.
The cell densities (unrounded data) of each Erlenmeyer flask were used to calculate the area under the growth curve (biomass integral). The biomass integral inhibition is given in Table 2 for each test concentration.
The inhibition(%) of the growth rate during the test is presented in Table 2.
Using linear interpolation, with mean measured concentrations on a logarithmic scale (base = 10), the 72h-EC50 for biomass integral and growth rate were 6.3 and 32 mg/L, respectively.
The No Observed Effect Concentration (NOEC), based on biomass integral at test termination, was determined with Williams' Test (Williams, 1972). Based on mean measured concentrations this revealed a NOEC of 1.0 mg F1EC per litre. However, the NOEC based on biological relevance (inhibition observed at > 10%) was 2.2 mg/L where only 2.6 % inhibition was observed.

Temperature. light intensity and pH:
The measured temperature of the thermostated incubator ranged between 22.6°C and 24.6°C, which is in agreement with the criterion of 21- 25°C controlled at ± 2°C. During the study five temperature registrations were higher than the range of 22.6 -24.6°C for a short period. These five temperature registrations were 24.9, 27.3, 26.7, 25.8 and 25.0°C, respectively. As the temperature increased only during a very short period, an effect on growth of the algae is not expected.
The light intensity at the level of the Erlenmeyer flasks was 75 μE/m2.s, which is in agreement with the criterion of 60 - 120 μE/m2.s (ISO, 1989).
The pH ranged between 7.5 and 8.5. According to OECD Guideline 201 the pH of the solutions should not increase more than 1.5 unit during the test.

DEVIATIONS FROM PROTOCOL:
The measured temperature of the thermostated incubator ranged between 22.6°C and 24.6°C, which is in agreement with the criterion of 21l - 25°C controlled at ± 2°C. During the study five temperature registrations were higher than the range of 22.6 - 24.6°C for a short period. These five temperature registrations were 24.9, 27.3, 26.7, 25.8 and 25.0°C, respectively. As the temperature increased only during a very short period, an effect on growth of the algae is not expected.
To the opinion of the study director these deviations did not influence the integrity of the study.
Results with reference substance (positive control):
From 4 - 7 April 2006 a reference test was conducted with potassium dichromate. The results of the test revealed an 72hEC50 for biomass integral and growth rate of 0.48 and 0.89 mg/l respectively. The results were in agreement with the results of an interlaboratory test (Hanstveit and Oldersma, 1981 ). The 72hEC50 for biomass integral should be in the range of 0.28 - 1.1 mg/l. The 72hEC50 for growth rate should be in the range of 0.14 - 1.6 mg/l.

 Table 1 : Mean measured concentration of F1EC in test solutions based on fluoride

 Nominal concentration of F1EC   

(mg/L)

 Measured concentration of F1EC based on fluoride (*)   

(mg/L)

 Mean measured concentration of F1EC (***)   

(mg/L)

 Day 0  Day 3
 0 (**)  -  -  0
 1  1.2 1.1   1.2 (120)
 2.2  2.1  2.6  2.4 (107)
 5.0  4.5  5.4  5.0 (100)
 11  10.3  11.3  10.8 (98)
 25  24.2  26.3  25.3 (101)

(*) The measured concentration fluoride originates from F1EC, however at the time of measurement F1EC itself was hydrolyzed.

(**) In the algal medium a small amount of fluoride was detected. As the amount was > 10% of the lowest test concentration, a correction for the background measured at 0 mg/L was used.

(***) Between brackets the percentage of nominal concentration is given.

 Table 2 : Mean measured algal cell densities during the test period, biomass integral inhibition and mean growth rate inhibition

 Nominal concentration   

(mg/L)

 mean cell density x 10E4 (*)

(cells/mL)

 Biomass integral inhibition on day 3

(%)

 Growth rate inhibition day 0 -3

(%)

 Day 1  Day 2  Day 3    
 0  3.274  26.74  138.4  -  -
 1.0  3.039  26.01  146.2  -3.1  -1.2
 2.2  2.980  25.59  121.2  10.4  2.6
 5.0  2.812  19.66  78.95  38.5  11
 11  3.307  10.22  20.99  77.7  38
 25  2.780  10.34  13.39  82.1  47

(*) On day 0 the cell density of the preculture was determined. Based on this result 0.353 mL of the preculture was added to Erlenmeyer flasks resulting in an initial concentration of 1.0 x 10E4 cells/mL.

Validity criteria fulfilled:
yes
Remarks:
The algal density in the control has increased at least 16 times after 3 days and pH has not increased more than 1.5 unit
Conclusions:
The 72h NOEC (biologically significant) and the 72h ErC50 of F1EC to algae were determined to be 2.2 and 32 mg/L based on the growth rate respectively.
Executive summary:

The toxicity of F1EC (MONOFLUOROETHYLENE CARBONATE) to algae (Selenastrum capricornutum) was tested according to the OECD GLP Principles to the OECD Guideline 201(OECD,1984).

Based on the results of a preliminary study, the study was conducted at nominal concentrations of 1.0, 2.2, 5.0, 11 and 25 mg/L. The fluoride content of the test solutions was measured with cuvette test LCK 323. Based on these results, the difference between the nominal and the mean measured concentrations was < 20% and for this reason the endpoints of the test were based on nominal concentrations.

The cell density increased 121 - 155 times in the controls, which meets the validity criterion of a minimal of 16 times increase. The pH of controls was 7.8 on day 0 and 8.5 on day 3 which is in accordance with the validity criterion of a maximum increase of 1.5 pH unit.

The cell density measurements were used to calculate the biomass integral and growth rate for each replicate. Using linear interpolation the 72h-EC50 for biomass integral and growth rate were 6.3 and 32 mg/L, respectively. The No Observed Effect Concentration (NOEC), based on biomass integral and growth rate at test termination, was determined to be 1 mg/L with Williams' Test (Williams, 1972). Based on biological significance/relevance (i.e. inhibition < 10%) the NOEC was 2.2 mg/L.

Description of key information

The 72h NOEC (biological significance) and the 72h ErC50 of the substance to algae were determined to be 2.2 and 32 mg/L based on the growth rate respectively.

There is no data available for marine algae species.

Key value for chemical safety assessment

EC50 for freshwater algae:
32 mg/L
EC10 or NOEC for freshwater algae:
2.2 mg/L

Additional information