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Short-term toxicity to fish

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Description of key information

In an acute flow-through test with L. macrochirus following a method similar to OECD TG 203 the 96-hour LC 50 of the test item was determined to be > 0.0065 mg Teflubenzuron/L (mean measured).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
Effect concentration:
> 0.006 mg/L

Additional information

A number of studies were submitted in the EU in accordance with Council Directive 91/414/EEC, which were summarized in the original Draft Assessment Report (DAR) Volume 3, B9 Ecotoxicology for teflubenzuron and its metabolites (2007). However, some of these studies were not considered reliable. Therefore, only for those studies that were accepted and considered reliable for use in the risk assessment in the original DAR, a summary is provided below.


Key information

Shell (1991): Teflubenzuron: 96 h acute toxicity to Lepomis macrochirus. unpublished report, report No. SBGR.90.211, according to Draft Assessment Report (2007) according to Council Directive 91/414/EEC, crossreference: MCA 8.2.1/02

A 96 h continuous-flow, acute toxicity test with bluegill sunfish (Lepomis macrochirus) was carried out following the OECD Guidelines for testing of chemicals, Guideline Number 9 (draft version, September 1982) and U.S. EPA Federal Register Part 797.1400 (1985). The test was performed with a single test substance concentration in the range of 0.0078 to 0.009 mg a.s./L which is close to the water solubility of approximately 0.010 mg a.s./L (instead of 100 mg a.s./L for a limit test). Stock solutions were prepared by dissolving 5g test substance in 800 mL acetone and thoroughly mixed with 3.5 kg of soda-glass balls (diameter of 1.5 to 2 mm). Air was passed through the balls to evaporate the aceton. The balls were than packed into a glass column (550 mm high, 80 mm diameter), fitted with a glass-slit sieve base. After preparation of the column mains dechlorinated water passed through from a > 200 L stock reservoir into both test vessels. To achieve a saturated solution of test item in the test vessels a system was set up, in which a stock reservoir (> 200 L) was passed through the apparatus into the test vessel and the overflows from these vessels were passed back into the 200 L reservoir. After four days the test item concentration was < 0.020 mg a.s./L. Immediately before test start the overflows from the test vessels were removed from the 200 L reservoir and diverted to run to waste.  Flow rate was 9.5 – 10.5 mL/min. Test item concentrations in the test vessels were measured on 4 days prior to the start of the definitive test, at the beginning of the test and at test termination, 96 hours later. All samples were extracted and analysed using a validated method.

The test vessels were glass aquaria (10 L) with overflows. 2 tanks were operated with test solution and 2 were operated as control. The experiment was carried out in a constant temperature room maintained at 22-23 °C, with a 16/8 hours light/dark cycle. Temperature, pH and oxygen content were measured during the test. Each tank contained 10 fish, i.e. 20 fish per treatment/control. The fish had a mean weight of 0.59 g and a mean length of 3.7 cm and were acclimated to test conditions 14 days before use. The fish were not fed during the test. At 3, 6, 24, 48, 72 and 96 hours, the fish were observed and the number of fish exhibiting toxic symptoms was recorded.

At the beginning of the test, pH averaged 7.7; pH decreased slightly at 48 h and increased to 7.85 at 96 hours. During the test dissolved oxygen concentration ranged from 8.0 to 8.2 (approx. 92% to 95% O2-satur.) in all test tanks. Temperature in the test tanks varied from 22 to 23 °C.

The concentrations of test item in the test vessels during 4 days prior to the start of the test fluctuated from 0.00078 to 0.0009 mg a.s./L. No consistent trend was observed through time. These concentrations are 78 to 90% of the reported water solubility of test item (approx. 0.01 mg a.s./L), but higher concentrations could not be achieved. At the beginning of the definitive test, the mean test item concentration was 0.0074 mg a.s./L. The mean concentration at the end of the test was 0.0055 mg a.s./L. Hence, the mean measured concentration was 0.0065 mg a.s./L. The mean decrease in concentration of test item in the test vessels over the duration of the test was 26%. Since no toxic effects were observed in either the control fish or the exposed fish the 96-hour LC50 was determined to be > 0.0065 mg a.s./L.

During the evaluation of teflubenzuron according to Council Directive 91/414/EEC, the 96-hour LC50 of > 0.0065 mg a.s./L was determined as key value for the risk assessment (please refer to the Draft Assessment Report for teflubenzuron, Volume 3 – B.8, 2007). 

The study was regarded acceptable for the hazard and risk assessment under Regulation (EC) No 1907/2006. 


Supporting information

The acute toxicity of the metabolites CL 902374 (CFPU, 3,5-dichloro-2,4-difluorophenyl­urea) and CL 907373 (CFA, 3,5-dichloro-2,4-difluoroaniline) to fish was investigated in 96-hour semi-static laboratory studies on rainbow trout (Onchorinchus mykiss) (Springborn Laboratories, 1998 and ECT, 2003).

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 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 Rain­bow Trout (Oncorhynchus mykiss) in a Static-Renewal Toxicity Test. Unpublished report No. ECO 97-302, according to Draft Assessment Report (2007) according to Council Directive 91/414/EEC, crossreference: MCA: 8.2.1/04

In the semi-static metabolite study of Springborn Laboratories, (1998) fish were exposed to a solvent control and to 5 nominal CFPU concentrations (0.5, 1, 2, 4 and 8 mg CFPU/L) in groups of 7 animals per treatment group (one replicate) in glass aquaria (14 L). Dimethylformamide (DMF; 0.1 mL/L = 0.01%) was used as a solvent. Test organisms were carefully transferred into freshly prepared test solutions at 24, 48 and 72 hours of exposure (renewal scheme) using a fine mesh net. In the test basins, temperature was adjusted to 14 °C ± 1°C for the 96 hour test. The light dark/cycle was 16/8 hours per day. The pH and oxygen content were measured during the test. The trout had an average weight of 1.7 g (range 1.3-2.5 g) and an average length of 5.4 cm (range 4.5-6.0 cm). Holding occurred at 13.5-15.2 °C. No mortality was observed during the 48-hour period prior to test initiation in the fish population used for the test. During the test, the basins were controlled at 24, 48 and 96 hours, and mortalities, as well as behavioural changes were recorded. During the definitive test, test solutions samples were taken for analytical verification, after mixing, from freshly prepared test solutions at hour 0, 24, 48, and 72 and 24-hour old test solutions at 96 hour to determine the concentrations of CL 902374 with a HPLC method.

During the study the pH ranged from 7.30 to 8.27 for the freshly prepared test solutions and from 7.05 to 7.87 for the 24-hour old test solutions. The dissolved oxygen concentrations ranged from 9.10 to 10.87 (88.3 to 105.4% of saturation) for the freshly prepared test solutions and from 8.51 to 9.98 (82.5 to 96.8% of saturation) for the 24-hour old test solutions. In the test solutions, the temperatures ranged from 13.9 to 14.7 °C for the freshly prepared test solutions and from 14.0 to 14.5 °C for the 24-hour old test solutions throughout the definitive study (daily measurements). The control aquarium temperature was 13.7 to 15.0 °C (continuous monitoring).

Concentrations of CFPU in samples of freshly prepared test solutions at 0, 24, 48, 72 hours averaged 98.4, 96.4, 99.3, and 100.6% of the nominal, respectively. The concentration of CL 902374 in test solutions was also verified at 96 hours in 24-hour old test solutions and averaged 99.8% of the nominal. Based on these results, the test concentrations were expressed as mean measured concentrations. The corresponding mean measured concentrations for the nominal concentrations of 0.495, 0.99, 1.98, 3.96 and 7.92 mg CFPU/L (considering the purity of test substance) were 0.5, 1.0, 2.0, 3.9 and 7.8 mg CFPU/L (0.5039, 0.9693, 1.965, 3.861, 7.776 mg CFPU/L) representing 101.8, 97.9, 99.2, 97.5 and 98.2% of the nominal, respectively.

During the exposure period, mortality occurred only at the highest mean measured test concentration (i.e. 7.8 mg CFPU/L). For this test concentration the cumulative mortality rate was 0% at 24 hours, 29% at 48 hours, 43% at 72 hours, and 43% at 96 hours. Sublethal effects (e.g. loss of equilibrium, lethargy, rapid respiration) were only observed at the mean measured test concentrations of 3.9 and 7.8 mg CFPU/L. No mortality or sublethal effects were observed for the controls, solvent control or at the mean measured test concentrations of 0.5, 1, and 2 mg CFU/L. The NOEC value was 2.0 mg CFU/L. The LC50, based on the highest treatment level, was higher than 7.8 mg CFPU/L.


ECT (2003): A Study on the Freshwater Fish (Rainbow Trout) Acute Toxicity of 3,5-dichloro-2,4-difluoroaniline. Unpublished report, report No. G4FB, according to Draft Assessment Report (2007) according to Council Directive 91/414/EEC, crossreference: MCA: 8.2.1/05

In the semi-static metabolite study of ECT (2003) fish were exposed to control and 5 nominal concentrations of 0.43, 0.94, 2.07, 4.55, 10 mg CFA/L. One replicate test vessel containing 7 fish was prepared per test item concentration and the control. The test was conducted in either 20 litre glass or 18 litre stainless steel tanks containing 10 L test solution at 15 ± 1°C. The test tanks were held on a 16/8 hours light/dark cycle and the light intensity was approximately 100-1,000 lux. During the test period the test solutions were renewed after 2 days. The number of dead fish was determined after 2-4 and 5-7 hours as 3 and 6 hours value, respectively and after 1, 2, 3 and 4 days after beginning of the test. To verify the nominally applied concentrations, samples were taken from the test solutions and analytically measured. Immediately after sampling and before shipment, the samples were stabilised and stored in glass bottles at -18 to –25°C. ECx values were calculated by Probit Analysis using Linear Maximum Likelihood Regression if applicable and NOEC and LOEC were reported additionally.

In the tanks, pH varied between 7.5 and 7.8 (SD 0.073) during the study. Temperature ranged from 15 to 16 °C (SD 0.306) and the oxygen concentration (% dissolved oxygen) varied from 79.1 to 99.3 % (SD 5.072). The analytically measured concentrations of the test item showed recoveries between 80.2 and 101.4% of the nominal concentrations on day 0 and day 2 of exposure, and between 79.2 and 93.4% on day 2 and day 4 of exposure (after renewal of test solutions after 2 days). The recovery for the stock solution was 98.0% at the beginning of the test, and 93.4% after renewal after 2 days. The mean total recovery for all test solutions was 89.3%. Since the average recovery of all test solutions was satisfactorily maintained within ± 20% of the nominal concentration throughout the study the bio­logical results were reported based on nominal concentrations.

No mortality was observed in the control and up to 4.55 mg CFA/L. Only at the highest tested concentration of 10 mg CFA/L all fish had died after 24 hours of exposure. Sublethal effects like impaired swimming behaviour and equilibrium occurred at 2.07 and 4.55 CFA/L. The EC50 (mortality, 96 h) was calculated to be 6.74 mg CFA/L based on nominal concentrations. The NOEC was estimated to be 4.55 mg CFA/L per expert judgement.

According to the EFSA conclusion of teflubenzuron (2008) the metabolites were evaluated as less toxic than the parent.



In an acute flow-through test with L. macrochirus following the OECD TG 203 (Shell, 1991) the 96-hour LC 50 of the test item was determined to be > 0.0065 mg a.s./L (mean measured). Supporting information of semi-static acute toxicity studies with the metabolites CL 902374 (CFPU) and CL 907373 (CFA) are available. In these studies with P. promelas the 96-hour LC50 values of the metabolites were > 7.8 mg CFPU/L (mean measured) and 6.74 mg CFA/L (nominal).