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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:
20-08-2002 to 22-08-2002
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Name: teflubenzuron
- CAS no.: 83121-18-0
Analytical monitoring:
yes
Details on sampling:
Samples of control, C1, C3 and C5 were taken at the start and the end of the test and analysed by HPLC/UV.
Vehicle:
yes
Remarks:
Aceton
Details on test solutions:
A stock solution (SL1) was prepared by dissolving the test item in Acetone. A second stock solution (SL2) was established by diluting SL1 in Medium M4. The test item concentrations were prepared by diluting SL2 with Medium M4 based on information provided by the sponsor and a pre-test.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: not specified
- Age of parental daphnids: 18 days
- Age at study initiation: 6 -24 h
- Source: not specified
- Feeding during test: none

ACCLIMATION
- Feeding frequency: 2 times per week with alage, TetraMin suspension
Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
48 h
Hardness:
not reported
Test temperature:
21.0 ± 0.05°C
pH:
7.57 - 8.20
Dissolved oxygen:
98.2 ± 3.6% corresponding to 8.7 ± 0.3 mg/L
Salinity:
not applicable
Conductivity:
not reported
Nominal and measured concentrations:
Nominal concentrations: control, 0.00088, 0.00188, 0.00413, 0.00909 and 0.02 mg/L
Measured concentrations: control, 0.00081, - , 0.00266, - , 0.02015 mg/L
Details on test conditions:
TEST CONDITIONS
- Test vessel: glass vessel, test volume 2 L
- Medium: dilution water “M4” (reconstituted water)
- Feeding: no feeding
- Aeration: no aeration
- Number of organisms: 20 larvae per concentration and control (four replicate test vessels each containing 5 larvae)
- Age at test initiation: young daphnids (6 - 24 h)
- Photoperiod: 16:8 hours light:dark
- Light intensity: ca. 221.2 lux
ASSESSED ENDPOINTS: 24 and 48 hours after the start of exposure, observations were made on daphnid immobility and on other signs of toxicity.
Reference substance (positive control):
not required
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
0.003 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: No
- Other biological observations: No
- Mortality of control: No
- Other adverse effects control: No
- Immobilisation of control: No
- Abnormal responses: No
- Any observations that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Statistical methods used to calculate ECx values was Probis analysis using Linear Max. Likelihood Regression.

The analytically determined concentrations of the test item showed recoveries between 78.9 and 98.7% of the nominal concentrations at the beginning of the test. The overall recovery based on the results of the stock solution and all test solution samples taken was 64.3–111.0% of the nominal test item concentra­tions. The average recovery based on the results of all test solutions was 87.4%.


 


Table 1:      Effects on Daphnia magna from teflubenzuron at different concentration levels at 0, 24 and 48 hours.




























































































































Concentration [mg/L]



Time [h]



Number of   mobile daphnids



Number of immobile daphnids



Immobil. [%]



Control



0



20



0



0



0.00088



20



0



0



0.00188



20



0



0



0.00413



20



0



0



0.00909



20



0



0



0.02



20



0



0



Control



24



20



0



0



0.00088



18



2



10



0.00188



13



7



35



0.00413



14



6



30



0.00909



5



15



75



0.02



8



12



60



Control



48



20



0



0



0.00088



17



3



15



0.00188



8



12



60



0.00413



12



8



40



0.00909



3



17



85



0.02



2



18



90



 

Validity criteria fulfilled:
yes

Description of key information

In a 48 h static acute toxicity study with Daphnia magna according to OECD 202, the EC50 of the test item was determined to be 0.0028 mg teflubenzuron/L based on mean measured concentration.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC50
Effect concentration:
0.003 mg/L

Additional information

One valid acute immobilisation test with the test item according to OECD 202 is available. Supportive information from acute immobilisations studys with the metabolites are summarized below, but not documented in separate IUCLID endpoint study records.


All presented studies were peer-reviewed during the assessment of teflubenzuron according to Council Directive 91/414/EEC.


Key information


ECT (2003): A Study on the Daphnia Acute Toxicity of BAS309 I (teflubenzuron), according to the EEC Directive 92/69 method C.2, „Acute Toxicity for Daphnia“. Unpublished report, report No. G3DA, according to Draft Assessment Report (2007) according to Council Directive 91/414/EEC, crossreference: MCA  8.2.4/01


To assess the acute toxicity of the test item to aquatic invertebrates a 48-hour semi-static immobility test with Daphnia magna was carried out according to OECD 202 in accordance with the GLP principles. A stock solution (SL1) was prepared by dissolving the test item in Acetone. A second stock solution (SL2) was established by diluting SL1 in Medium M4. The test item concentrations were prepared by diluting SL2 with Medium M4 based on information provided by the sponsor and a pre-test. The dose levels applied in the test were exposed to a control, 0.00088, 0.00188, 0.00413, 0.00909 and 0.02 mg a.s./L nominal concentrations. One day before starting the test all young daphnids were removed from holding vessels containing adults. At least 6 hours before placing young daphnids into the test vessels, the new borne daphnids were separated from parental daph­nids (18 days old). After temperature adaptation, the young daphnids were transferred into the test solutions with a glass pipette by a randomizing proce­dure. 4 replicates of each concentration and the control were established con­taining 5 daphnids each. The glass covered test vessels were 300 mL glass vessels containing 200 mL test solution. The photoperiod was 16/8 hours light/dark with a light intensity of 221 ± 24 lx. There was no aera­tion and no feeding during the test. To verify the nominally applied concentrations, samples were taken at the start and the end of the test and analysed by HPLC/UV. The statistical method used to calculate the ECx values was Probit analysis using Linear Max. Likelihood Regression.


The temperature was 21.0 ± 0.05°C inside the test chamber and 20.8 ± 0.11°C outside the test chamber. The pH-values ranged between 7.57 and 8.20 and the oxygen concentration was 98.2 ± 3.6% corresponding to 8.7 ± 0.3 mg/L. The analytically determined concentrations of the test item showed recoveries between 78.9 and 98.7% of the nominal concentrations at the beginning of the test. The overall recovery based on the results of the stock solution and all test solution samples taken was 64.3–111.0% of the nominal test item concentra­tions. The average recovery based on the results of all test solutions was 87.4%. No immobilisation was observed throughout the test in the control. 15 % of the daphnids were immobile at the lowest treatment group after 48 h of exposure. Nevertheless, no statistically significant difference between the lowest treatment and the control was determined. Therefore a NOEC of 0.00088 mg a.s./L was established based on nominal concentration, which corresponds to 0.00081 mg a.s./L based on measured concentrations . The 48-h EC50 was determined to be 0.0028 mg a.s./L (95% confidence limits, lower/upper: 0.0019 / 0.0043 mg a.s./L), based on measured concentrations. Since all validity criteria of the OECD 202 were met, the study is considered acceptable for the use in the risk assessment.


In the Draft Assessment Report for teflubezuron according to  Council Directive 91/414/EEC (2007) the 48-h EC50 of 0.0028 mg a.s./L  is indicated as key value for the short-term toxicity of teflubenzuron to aquatic invertebrates. 


 


Supporting Information


The acute toxicity of the teflubenzuron metabolites CL 902374 (CFPU, 3,5-dichloro-2,4-difluorophenyl­urea) and CL 907373 (CFA, 3,5-dichloro-2,4-difluoroaniline) to aquatic invertebrates was investigated in 48-hour semi-static laboratory studies on Daphnia magna.


For the supporting information no separate IUCLID endpoint study records have been prepared. For details please refer to the original Draft Assessment Report (DAR) Volume 3 Ecotoxicology and the Draft Renewal Assessment Report for teflubenzuron according to the Council Directive 91/414/EEC, Volume 3 – B.9 (AS) (2007).


Springborn Laboratories (1998): Acute Toxicity of CL 902374 to Daph­nia magna in a Static-Renewal Toxicity Test. Unpublished report, report No.: ECO 97-303; according to Draft Assessment Report (2007) according to Council Directive 91/414/EEC, crossreference: MCA 8.2.4/03


In the semi-static metabolite study daphnids were exposed to a solvent control and to 5 nominal CL 902374 (CFPU) concentrations of 10, 18, 32, 56 and 100 mg CFPU/L. Stock and test solutions were prepared at 0 and 24 hours. The stock solutions were prepared by adding the test item to Dimethylformamide (DMF), which was used as solvent. DMF was added to the test medium at a concentration of 1 mL/L (0.1% v/v). Four test vessels (250 mL flasks containing 200 mL test solution) containing five daphnids for each concentration were placed in a water bath at 19.7 – 19.9°C. For the analytical verification samples were taken from freshly prepared (0 and 24 h) and from old (24 h and 48h) test solutions and analysed by HPLC/UV. The numbers of immobilised daphnids, biological observations and observa­tions on the physical characteristics of each replicate were recorded after 0, 24 and 48 hours. The temperature of the water bath was continuously monitored. A computer programme modified from the programme of Stephan (Peltier and Weber, 1985) was used to calculate the EC50 values and the 95% confidence intervals. For the observed sublethal effects the NOEC was determined using one-way analysis of variance, Williams test or Dunnett’s test.


The pH ranged between 7.5 to 7.91, the dissolved oxygen between 8.44 and 8.88 mg/L (93 and 98%, respectively). The temperature in the test solutions and in the water bath was 19.7 to 19.9 °C. The total hardness in the control and in the highest concentration was 228 and 222 mg/L as CaCO3, respectively. Alka­linity was 600 µSiemens/cm for control and highest concentration. At the test concentration of 10 mg CFPU/L analytical recoveries reached 91.1 to 95.8% of the nominal but recoveries were highly variable for test concentrations higher than 10 mg CFPU/L (32.1 to 94.9% of the nominal). Therefore the biological results were reported based on mean measured concentrations.


The 48-hour EC50 for CFPU to Daphnia magna was calculated to be 15.1 mg CFPU/L, with a 95% confidence interval of 12.6 to 18.1 mg CFPU/L (mean measured). The NOEC was estimated to be < 6.4 mg CFPU/L.


 


ECT (2003): A Study on the Daphnia Acute Toxicity of 3,5-dichloro-2,4-difluoroaniline. Unpublished report No. G4DA , according to Draft Assessment Report (2007) according to Council Directive 91/414/EEC, crossreference: MCA 8.2.4/04


In the static metabolite study of ECT (2003) daphnids were exposed to control and 5 nominal CL 907373 (CFA) concentrations of 11.0, 5.5, 2.75, 1.375, 0.6875 mg CFA/L. A stock solution of CFA was prepared by dissolving 33.0 mg of the test item in 3 L Medium M4. After the stock solution was shaken, ultra-sonificated and stirred the test concentrations were prepared by dilution with Medium M4. The test vessels (glass beakers containing 150 – 200 mL test solution) were placed into a temperature controlled room at 20°C. The daphnids were held in a specific medium (Medium M4) under conditions similar to the testing conditions. The daphnids were fed with TetraMin and algae. The offspring used for the test derived from daphnids with the age of 15 to 45 days. At test start 5 daphnids aged 6 to 24 hours were added to the test vessels. For each concentration level and control 4 replicates were used. The daphnids were not fed during the test period. For the analytical verification samples were taken from freshly prepared (0 and 24 h) and from old (24 h and 48h) test solutions and analysed by HPLC/UV. The numbers of immobilised daphnids, biological observations and observa­tions on the physical characteristics of each replicate were recorded after 0, 24 and 48 hours. The statistical method used to calculate the ECx values was Probit Analysis using Linear Maximum Likelihood Regression.


In the test vessels, pH varied between 7.6 and 7.6 (SD 0.14), temperature ranged from 20.5 to 21.1 °C (SD 0.21) and the oxygen concentration (% dissolved oxygen) varied from 95.9 to 107.2 % (SD 3.7). The analytically determined concentrations of the test item showed recoveries between 79.0 and 98.6% of the nominal concentrations at the beginning of the test. The recovery rate for the nominal test item concentrations after the expo­sure period of 48 h varied between 64.1 and 100.6%. The mean total recovery for all test solutions was 87.4%. A correction of the bio­logical results based on measured concentrations was not performed, because the average recovery of all test solutions was satisfactorily maintained within ± 20% of the nominal concentration throughout the test.


No immobilisation was observed in the control and the lowest test concentration of 0.6875 mg CFA/L. At 2.75 mg CFA/L 9 immobile daphnids were observed after 48 h of exposure. The EC50 (immobilisation, 48 h) was calculated to be 1.48 mg CFA/L based on nominal concentrations.


 


Conclusion


In an acute semi-static test with D. magna according to OECD TG 202 the 48-hour EC 50 of the test item was determined to be 0.0028 mg a.s./L (measured). Supporting information of acute toxicity studies with the metabolites of the test item CL 902374 (CFPU) and CL 907373 (CFA) are available. In these studies with D. magna the 48-hour EC50 values of CFU was 15.1 mg CFPU/L (mean measured) and 1.48 mg CFA/L (nominal). Based on the most reliable study on the active substance the 48-hour EC50 is considered to be 0.0028 mg a.s./L. This value is fully in line with the conclusion drawn in the Draft Assessment Report for teflubenzuron prepared according to the Council Directive 91/414/EEC, Volume 3 – B.9 (AS), 2007.