2-chloro-N-[[[4-(trifluoromethoxy)phenyl]amino]carbonyl]benzamide

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation/completion: 26 October 2001 to 04 April 2002.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 72-4 (Fish Early Life-Stage and Aquatic Invertebrate Life-Cycle Studies)

Version / remarks:

EPA-FIFRA

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The nominal concentrations were 0 (control, solvent control), and 25 µg test item/L.

Sampling method:

Water quality parameters: Temperature, dissolved oxygen, and pH were measured in each aquarium on days 0, 7, 14, 19, 26, 32, and 36. The temperature was measured with a single point thermometer and a Min./Max. thermometer for continuous temperature monitoring. The dissolved oxygen concentration was measured with a dissolved oxygen meter and probe (WTW Oxi 325). The pH was measured using a combined glass electrode connected to a pH meter (WTW pH 323). Hardness and alkalinity were measured in the media from a control vessel and a vessel containing the highest concentration of test item at least weekly. Total hardness was measured by the EDTA titrimetric method and total alkalinity was determined by potentiometric titration to an endpoint
of pH 4.5.

Biological observations: Eggs: On day 1, 2, and 3 following start of exposure, each egg cup was examined. The number of live, dead, and unaccounted for eggs was recorded and the dead eggs were discarded. Dead eggs were identified by a marked loss of translucency and change in coloration. The criteria for embryo death was the absence of body
movement and/or absence of heart-beat. After hatching has begun, the egg cups were not handled in order to avoid possible physical damage to the newly hatched fry. Only dead eggs and fry were accounted for and removed at this time. Start of hatching and end of hatching (about 90 % or more of the living embryos from the control group hatched; day 0 post-hatch) was considered as two distinct entities.

After hatch was complete (day 4), the number of live, deformed, dead, and unaccounted for fry was recorded from each egg cup. Percentage hatch was calculated as the number of live fry in each egg cup/60 eggs.
Following the completion of hatch, daily observations were recorded on

1. fry mortality
2. sublethal effects (lethargy and gross abnormalities in behaviour, e.g.,
loss of equilibrium, respiratory function, exophthalmus, feeding activity,
reaction to external stimuli, and physical appearance, e.g., deformities and
coloration).

The criteria for death of larvae and juvenile fish were:

1. immobility and/or
2. absence of respiratory movement and/or
3. absence of heart-beat and/or
4. white opaque colouration of the central nervous system and/or
5. lack of reaction to mechanical stimulus.

A thorough search for dead fry was made daily in all aquaria. The number of live fry in each aquarium was estimated each day throughout the test. These counts are only estimations due to the difficulty in observing the very small, mobile fry. Positive counts of eggs or fish were performed at the start of exposure, when the fry were released into (day 0 post-hatch) and from the larval chambers (day 10 post-hatch), and on day 32 post-hatch. On day 32 post-hatch after sacrificing the fish, individual total lengths and individual wet weights of the fish were measured with a
calliper to the nearest 0.01 mm. Individual wet weight (blotted dry) was determined to the nearest 0.1 mg. The fish were dried at 60 °C overnight and the dry weight was determined to the nearest 0.1 mg.

Vehicle:

yes

Remarks:

DMF (0.1 mL/L in the test vessels)

Details on test solutions:

A constant flow test item delivery system equipped with syringe pumps for solvent and test item stock solution delivery and membrane pumps for dilution water delivery was employed to maintain a control, a solvent control with 0.1 ml DMF/L and a test concentration of 25 µg test item /L with 0.1 ml DMF/L in the test vessels. The treated test solution and the controls were set up in duplicate. All parts of the dosing system
which came into contact with the test item were constructed entirely out of Teflon in order to prevent adsorption of the test item. The solvent and test item stock solution were added directly into the dilution water stream.
The flow rates of stock solution and dilution water were calculated to result in the desired nominal concentrations with a test solution flow rate of 50 ml per minute for each test vessel. The nominal concentration of the test item stock solution was 250 mg test item /L. The flow rate of the dilution water and the stock solutions was adjusted so that the resulting
concentrations were 25 µg test item/L and 0.1 mL DMF/L. Prior to start of the exposure and frequently during the test, the flow rates were controlled for proper delivery rates. The flow rate for each aquarium was 50 mL/min resulting in 7.2 volume exchanges per day for the 10 L test solution volume.

Test organisms (species):

Pimephales promelas

Details on test organisms:

Fathead minnow (Pimephales promelas) was selected as the test organism since it is recommended by the OECD (1992) and is commonly used in chronic freshwater toxicity studies. Characteristics which make fathead minnow suitable for early life-stage toxicity studies are their ease of handling, their known sensitivity to a variety of toxicants, the ready availability of eggs, and the extensive historical database for this common fish species. The eggs were introduced into the test solutions within 24 hours after spawning.

Test type:

flow-through

Water media type:

freshwater

Remarks:

deionized and subsequently reconstituted Horn well water.

Limit test:

yes

Total exposure duration:

36 d

Hardness:

148 to 180 mg/L CaCO3

Test temperature:

24.3 to 25.6°C

pH:

range of 6.88 to 7.73

Dissolved oxygen:

range of 80 to 111% of the air-saturation value

Nominal and measured concentrations:

Nominal concentration: 25 µg/L. mean measured concentration: 22.8 µg/L.

Details on test conditions:

The aquaria were impartially positioned in a waterbath containing circulating water designed to maintain the test solution temperatures at 25 ± 1 °C. The aquaria had the following dimensions: length: 29.5 cm, width: 23.5 cm and were fabricated of stainless steel. The water depth was approximately 16 cm resulting in a volume of approximately 10 litres test solution. The aquaria were labelled with test concentration, replicate identification, and study number. Illumination was provided by fluorescent bulbs located above the test aquaria. A 16 hours light and 8 hours
dark photoperiod with a 30 minutes transition period, controlled by an automatic timer, was maintained throughout the test. The light intensity was measured with a Lutron LX-105 light meter at the test solutions surface during the light period and ranged from 400 - 800 Lux.

Key result

Duration:

36 d

Dose descriptor:

NOEC

Effect conc.:

>= 22.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: all parameters

Key result

Duration:

36 d

Dose descriptor:

LOEC

Effect conc.:

> 22.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: all parameters

Details on results:

Mortality: There was no effect of the test item at the start and end of hatching.

Sublethal effects: No negative statistically significant effect on egg hatchability, larval development, behaviour, growth, and survival.

Reported statistics and error estimates:

The mean measured concentrations of the active substance and the corresponding biological response data (egg mortality, egg hatchability, total individual length, wet and dry weight, fish showing abnormal behaviour, number of fish with deformations, and total mortality) were used to determine the NOEC, and the LOEC. Analyses were performed using the mean organism response in each treatment group rather
than individual response values.

All statistical analyses were conducted at the 95% level of certainty except in the case of Shapiro-Wilks Tests, and Bartlett's Tests, in which the 99% level of certainty was applied. The 99% level of certainty is preferred for qualifying tests. The following procedures were used:

1) Significant differences in the percent egg hatchability and survival were
determined after transformation (e.g., arcsine square-root percentage) of the data.

2) As a check on the assumption of normality distribution and homogeneity of variance, implicit in parametric statistics, data for each endpoint were analysed using Shapiro Wilk's test for normality (Weber et al. 1989) and Bartlett's test (Horning and Weber, 1985).

3) For each endpoint, the performance of organisms exposed to each treatment level of the test substance was compared with the performance of the control using the Bonferroni t-test (Weber et al. 1989) since the degrees of freedom were to low to perform the Dunnetts or Williams test.

The results of egg hatchability, survival, length, wet and dry weight of Feathed minnow larvae during 36 days of exposure to the test item are shown in Table 1.

 

Table 1 - Fish early life stage toxicity test; egg hatchability, survival, length, wet and dry weight of Fathead minnow exposed to SIR 8514 (*figures represent Mean measured Test Concentration in µg/ L)

Treatment	Replicate	Hatching success on completion of hatch (day 0 posthatch) [%]*	Post-hatch survival until day 10 post-hatch [%]*	Post-hatch survival until day 32 posthatch [%]*	Total Length (s.d.) on day 32 posthatch [mm]*	Wet weight (s.d.) on day 32 posthatch [mg/fish]*	Dry weight (s.d.) on day 32 posthatch [mg/fish]*
Control	A	85	95	95	27(±4)	195(±66)	47(±16)
	B	87	88	85	27(±3)	191(±59)	45(±16)
	Mean	86	91	90	27(±3)	193(±62)	46(±16)
Solvent Control	A	82	90	90	27(±2)	200(±67)	48(±17)
	B	87	95	95	26(±4)	187(±81)	44(±20)
	Mean	84	93	93	27(±3)	193(±74)	46(±18)
22.8*	A	87	100	100	26(±4)	173(±68)	42(±17)
	B	78	90	88	27(±4)	184(±76)	44(±19)
	Mean	83	95	94	26(±4)	178(±72)	43(±18)

 

Tested parameters for FELS study

• First Hatch (study day 3)


• Hatching Rate (study day 4)


• Time to Completion of Hatch (study day 4)


• Larval deformities (study day 14)


• Larval Survival (study day 14)


• Behavioural changes and deformations (study day 36)


• Post Hatch Survival (study day 36)


• Length (study day 36)


• Wet Weight (study day 36)


• Dry Weight (study day 36)

Validity criteria fulfilled:

yes

Conclusions:

The test item had no negative effect on the early life stages of fathead minnow (Pimephales promelas) within the practical limit of its water solubility under conditions of testing.

Executive summary:

The study was conducted at the nominal test concentration of 25 µg test item/L as a limit test at the practical limit of the water solubility. The 36 days exposure started with fathead minnow eggs <24 hours old. The mean measured concentration was 22.8 µg a.s. /L. Analytical measurements were within ± 20 % of the mean measured concentration. All results are based on the mean measured active substance (a.s.) concentrations.

The NOEC, and LOEC for the following endpoints: first hatch (Study day 3), hatching rate (study day 4), time to completion of hatch (study day 4), larval deformities (study day 14), larval survival, behavioural changes and deformations (study day 36), post Hatch Survival (study day 36), length (study day 36), wet weight (study day 36), dry Weight (study day 36).

Description of key information

 The test item had no effect on the early life stages of fathead minnow (Pimephales promelas) within the practical limit of its water solubility under the conditions of testing. The NOEC for all tested parameters is >= 22.8 µg/L and the LOEC is > 22.8 µg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

>= 22.8 µg/L