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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The water fleas were exposed to the test substance from 14 - 16 September 2005. The test solutions were analysed on 15 and 16 September 2005 (GC/FID) and on 10 October 2005 (fluoride).
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
See details on analytical methods
Vehicle:
no
Details on test solutions:
Based on a preliminary test the study was conducted at F1EC concentrations of 0, 1.0, 2.2, 5.0, 11 and 25 mg/L.
The test solutions were renewed after 24 hours. On the day of test initiation and after 24 hours a stock solution was prepared by dissolving 100 mg (66.7 μL) F1EC in 500 mL Elendt M4 medium resulting in an FlEC concentration of 200 mg/L. Bulk solutions were prepared at test initiation and after 24 hours by dilution of the bulk solution in ELENDT M4. With these solutions three glass beakers were filled for each concentration with 200 mL bulk solution. Three control glass beakers were prepared. The water fleas were exposed to the test solutions during 48 hours. All test solutions were clear and no undissolved test substance was visible.
Test organisms (species):
Daphnia magna
Details on test organisms:
Water fleas (Daphnia magna, Crustacea) used in this test originate from a culture received on 4 February 2003 from Aquasense b.v., Amsterdam, The Netherlands. The water fleas are in culture since 4 February 2003. Water fleas of the same age are cu!tured in vessels with 2 liters Elendt M4 medium. They are fed with an algal suspension of Chlorella vulgaris. ·
The water fleas are cultured in a climate chamber with a temperature setpoint of 20°C. The light regime is 16 hours light and 8 hours darkness per day. The light is switched on at 6 a.m. Each week a new culture of water
fleas is started and water fteas older than 2 weeks are removed. The age of the water fleas used in the test, described in this report, was 0 - 24 hours at the start of the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
No data
Test temperature:
Measured to range from 20.2 to 22°C.
pH:
At test initiation the pH of the Elendt medium was 8.1, the pH in the highest test concentration was 7.3.
After 24 hours test solutions were renewed. The pH of the Elendt medium was 8.1.
Dissolved oxygen:
At test initiation the oxygen content of the Elendt medium was 8.8 mg/l, the oxygen content in the highest test concentration was 8.8mg/L.
After 24 hours test solutions were renewed. The oxygen content of the Elendt medium was 8.7 mg/L.
At test termination the oxygen content of the test solutions was > 5.6 mg/L
Nominal and measured concentrations:
See table 2 in the result field.
Details on test conditions:
Test initiation and environmental conditions:
Ten water fleas were added to each glass beaker. The glass beakers were placed in a climate chamber with a temperature setpoint of 20°C. The air temperature was recorded once every hour. The light regime was 16 hours light and 8 hours dark (light on from 6 a.m. to 10 p.m.).

Biological observations and measurements :
The number of mobile and immobile water fleas and the number of water fleas showing sublethal effects was recorded 24 and 4.8 hours after test initiation. After 24 hours immobile water fleas were removed from the test solutions. Immobile water fleas are those animals which are not able to swim within 15 seconds after gentle agitation of the glass beaker (OECD, 2004). ·
At test initiation the pH, temperature and oxygen content was measured in the Elendt medium and in the highest test concentration. At test termination the pH, temperature and oxygen content were measured in one glass
beaker per test concentration.
Reference substance (positive control):
yes
Remarks:
potassium dichromate was tested in March 2015, see the field on results.
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
11.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
8.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other:
Remarks:
95% CI 7.1 - 10 mg/L.
Key result
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
2.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Biological observations :
No immobility or sublethal effects were observed in the control glass beakers.
The biological results of the test are presented in Table 1.
The No Observed Effect Concentration (NOEC) based on immobility and nominal concentrations was determined with Fisher's Exact Test (Sokal and Rohlf, 1981) and was equal to 2.2 mg/L.
The 24h and 48h EC50 based on nominal concentrations were calculated with probit analysis and were 11.5 mg/l and 8.4 mg/L respectively.

Analysis of F1EC based on fluoride:
The results of the analysis with the cuvette test is summarised in Table 2. Samples with a concentration of 11 and 25 mg/L were diluted 2 and 4 times, respectively with Elendt M4 medium before analysis.
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.

Test chamber :
The measured temperature of the thermostated climate chamber ranged between 20.0°C and 20.8°C. During the study there was a power failure of the climate chamber and for this reason the temperature ranged from 18.5 to 22.4°C during a period of 9 hours which is not in agreement with the criterion of 18 - 22°C controlled at ± 1°C. As this temperature was only slightly higher than the recommended temperature range, this deviation did not influence the results of the study.
Results with reference substance (positive control):
On a regular basis an acute toxicity test with water fleas and the reference substance potassium dichromate is conducted. The most recent test was conducted from 2 - 3 March 2005 (De Groot, 2005). The results revealed an
24hEC50 of 1.18 mg/L with a 95% confidence interval of 1.11 - 1.25 mg/L. According to ISO 6341 (1996) the 24hEC50 of potassium dichromate should be within the range of 0.6 - 1.7 mg/L.
Reported statistics and error estimates:
The most important endpoint for the study is immobility. The NOEC for immobility was determined with Fisher's Exact Test. The EC50 for immobility was determined with probit analysis. To verify the correct working of the probit analysis, a standard data set was used.

The output from the probit procedure which was used in our Department corresponded with the output from the standard data set. For this reason it can be concluded that the procedure was valid .
  Table 1 : Immobility of water fteas during the test

Nominal concentration

(mg/L)

 Number of mobile water fleas      

 Mean immobility at test termination

(%)

   0 hours  24 hours (*)  48 hours  
 0  10  10  10  
 0  10  10  10  0
 0  10  10  10  
 1.0  10  10  10  
 1.0  10  10  10  0
 1.0  10  10  10

 

 2.2  10  10  10  
 2.2  10  10  10  0
 2.2  10  10  10  
 5.0  10  10  9  
 5.0  10  10  9  13
 5.0  10  10 (2)  8  
 11  10  8  4  
 11  10  7  3  70
 11  10  5  2  
 25  10  0  0  
 25  10  0  0  100
 25  10  0  0  

(*) Between brackets the number of water fleas showing sublethal effects is given, sublethal effects were less activity.

 Table 2: Mean measured concentration of F1EC in test solutions based on fluoride

Nominal concentration F1EC  Measured concentration F1EC based on fluoride (mg/L) (1)     Mean measured concentration F1EC (mg/L) (3)
   Day 1  Day 2

 

 0 (2)  -  -

 0

 1.0  1.1  1.2  1.2 (120)
 2.2  2.6  2.6  2.6 (118)
 5.0  5.9  5.7  5.8 (116)
 11  11.9  11.7  11.8 (107)
 25  25.3  25.6  25.5 (102)

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

(2) In the Elendt M4 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.

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

  Table 3: Measured water parameters of test solutions at test termination
 Nominal concentration (mg/L)  Temperature (°C)  Oxygen content (mg/L)  pH
 0  20.3  8.8  8.2
 1.0  20.2  8.7  8
 2.2  20.2  8.7  7.9
 5.0  20.2  8.6  7.8
 11  20.2  8.6  7.7
 25 (*)  22.0  8.6  7.4

(*) mean value of the three glass beakers. The water parameters in the highest test concentration were measured after 24 hours in three glass beakers because 100% mortality was observed

Validity criteria fulfilled:
yes
Remarks:
There was no immobilisation in the control (< 10%), the dissolved O2 at the end of the test was > 3 mg/l in control and test vessels.
Conclusions:
The 48h EC50 of F1EC to daphnia magna was determined to be 8.4 mg/L.
Executive summary:

The acute toxicity of F1EC to water fleas (Daphnia magna) was tested following OECD Guideline 202 (OECD, 2004) and according to GLP.

Based on the results of a preliminary study, the study was conducted at nominal F1EC concentrations of 0, 1.0, 2.2, 5.0, 11 and 25 mg/L. Three glass beakers, each with 0.20 liter test solution, were prepared per concentration and 10 water fleas were added to each glass beaker. The test solutions were renewed after 24 hours. Water fleas were exposed for 48 hours to the test solutions.

To study the exposure of the daphnids fo F1EC, samples of the test solutions were taken before and after renewal and analyzed with GC/FID. To check the linearity of the F1EC rneasurement, calibration samples were prepared. The results showed that the correlation coefficient of the calibration line was > 0.99 and linear regression was used to determine the concentration F1EC in the samples. Even at test initiation the test compound was almost completely hydrolyzed. For this reason valid results to characterise the exposure of the daphnids were not obtained with GC analysis and it was decided to determine the concentration F1EC based on the fluoride content of the samples with cuvette test LCK 323. F1EC contains 18% fluoride based on the molecular formula. Based on the results obtained, 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.

No immobility was observed in the control glass beakers. The No Observed Effect Concentration (NOEC) based on immobility and nominal concentrations was determined with Fisher's Exact Test and was equal to 2.2 mg/L. The 24h-EC50 and 48h-EC50 based on nominal concentrations were calculated with probit analysis and were l l.5 mg/L and 8.4 mg/L, respectively.

Description of key information

The acute toxicity of the substance to water fleas (Daphnia magna) was tested following OECD Guideline 202 (OECD, 2004) and according to GLP.

No immobility was observed in the control glass beakers. The No Observed Effect Concentration (NOEC) based on immobility and nominal concentrations was equal to 2.2 mg/L. The 48h-EC50 based on nominal concentrations was 8.4 mg/L.

No data on marine invertebrate species is available.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
8.4 mg/L

Additional information