N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish life cycle toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Dec 2001 to 8 Jan 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 72-5 (Fish Life Cycle Toxicity)

Version / remarks:

1986

Qualifier:

according to guideline

Guideline:

other: Fish Life Cycle Toxicity Test, 540/9-86-137

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples of each test concentration and the controls, as well as of the stock solution were taken in weekly intervals for the determination of the actual concentrations of the test item during the whole period of exposure. At the start and end of an exposure period (F0 - F1 generation), samples were taken in all replicates. During the remaining period, samples were taken in alternate replicates. In addition, specimens were taken before day 0 to check the diluter system. Samples were taken subsurface from the center of the test chambers. The results from each sampling day were used to calculate the mean measured concentrations of the test item at each exposure level. The samples were stored in 250 mL amber glass bottles. Storage stability was checked for a period of 419 days. Samples which could not be analysed immediately were frozen and kept at -18°C to —22°C until analysis.

Vehicle:

yes

Remarks:

Triethylene glicol

Details on test solutions:

Stock solutions of a targeted concentration of 437.2 and 302.7 mg/L were prepared for the diluter of the F0-generation and of 437.2 mg/L for the diluter of the F 1-generation. Different volumes of stock solution were prepared depending on the time period the stock should last:

F0 Generation:
- 109.6 mg test item were with and up to 250 mL with triethylene glycol on day -1
- 218.6-219.0 mg test item were mixed with and made up to 500 mL with triethylene glycol on day 23, 31, 40, 48, 71.
- 437.3-437.9 mg test item were mixed with and made up to 1000 mL with triethylene glycol on day 53, 62, 75, 85, 94, 105, 114, 123, 132, 141, 148, 159, 167, 177, 186, 195, 203, 213, 221, 230,239, 248, 257, 266, 275, 283

F1 Generation:
- 437.2-437.6 mg test item were mixed with and made up to 1000 mL with triethylene glycol on day 160, 180, 193, 210, 220, 230, 239, 249, 259, 269, 279, 289, 298, 320.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
The eggs arrived at the test facility within approximately 5 hours after collection from the breeding tiles. The eggs did not receive any disinfection or medical treatment.

POST-HATCH FEEDING
The food used during the study included commercially prepared Tetramin flakes and brine shrimp nauplii (Artemia salina). The fry were fed live brine shrimp nauplii starting on day 0 post-hatch and additionally Tetramin starting on day 17 post-hatch (fry were fed ad libitum). The fish were fed twice daily from Monday to Sunday (morning and afternoon).

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

293 d

Remarks on exposure duration:

The total exposure duration of the FO-generation was 298 days and 61 days of the F1- generation.

Hardness:

F0: 148 to 196 mg CaCO3/L
F1: 167 to 205 mg CaCO3/L

Test temperature:

F0: 24.2 to 25.8 °C
F1: 24.0 to 25.7°C

pH:

F0: 7.7 to 8.7
F1: 8.0 to 8.5

Dissolved oxygen:

F0 & F1: 5.7 to 9.2 mg/L

Conductivity:

F0: 367 to 420 µS/cm
F1: 369 to 408 µS/cm

Nominal and measured concentrations:

Nominal concentrations: 0.0025, 0.0050, 0.010, 0.020, 0.040 mg/L
Measured concentrations:
- F0: 0.0031 ± 0.001, 0.0072 ± 0.0023, 0.013 ± 0.0041, 0.024 ± 0.0062, 0.042 ± 0.0097 mg/L
- F1: 0.0029 ± 0.0007, 0.0057 ± 0.0019, 0.010 ± 0.0030, 0.021 ± 0.0057, 0.045 mg/L

Details on test conditions:

TEST SYSTEM
- Emybro cups: Egg incubation cups (-5 to 0 dph), 4 x 10 L/concentration; 7 cm inner diameter x 8 cm high containers made out of a glass cylinder with a bottom of stainless steel screen (0.36 mm width of mesh, 0.2 mm thickness of wire).
- Test vessel: Glass aquaria of 12 litres were employed, yielding 10 litres of replicate tank volume (30/20/20 cm; depth of the water: 17cm)
- Aeration: During the initial phase of day 5 to 15, the test solutions were aerated during the night when the flow of test solution / water was stopped due to clogging of the siphons and the danger of an
overow and hence of loss of fish. From day 64 to the end of the exposure, aeration was necessary to maintain the dissolved oxygen at acceptable levels.
- Type of flow-through: proportional diluter
- Renewal rate of test solution: between 1920 and 3520 L/24h during the F0- and between 960 and 2880 L/24h during the F1-generation
- No. of fertilized eggs/embryos per vessel: 44
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: well water; prior to its use, the water passed through an activated carbon filter and a UV-sterilizer.
- Particulate matter: 0 mg/L
- Intervals of water quality measurement: every 6 months

OTHER TEST CONDITIONS
- Photoperiod: The test was conducted with a varying photoperiod, which simulated the dawn and dusk times of Evansville, Indiana as indicated by Benoit, 198. At day 5 (0 dph), the day length was adjusted to be as for 1 December (Evansville). The photoperiod was then adjusted accordingly during the duration of the F0 adult stage until day 201 (196 dph), when the maximum day length was attained. The maximum day length was kept until the end of exposure to maximise egg production. For the whole of the F1-generation, the photoperiod was kept at maximum day length.
- Light intensity: for the F0-generation measured at the water surface was 586 Lux ± 89 on day -27, 568 Lux ± 95 on day 57, and 604 Lux ± 89 on day 287. The corresponding values for the F1-generation were 609 Lux ± 110 on day 187.

EFFECT PARAMETERS MEASURED:
Abnormal (sublethal) behavioural or physical changes (loss of equilibrium, erratic swimming, loss of reex, excitability, discoloration, or change in appearance or behaviour) and mortality were monitored by visually inspecting each chamber on week days and recording the data. Dead test organisms were removed when first observed. On day 56 post-hatch, all surviving fish were photographed to obtain measurement of length.
F0: On day 162 and 293 post-hatch, adults were sacrificed and measured for total length, blotted on paper towels to remove excess moisture and weighed by analytical balance. In addition, the gonads were dissected and weighed to determine the gonadosomatic index (GSD). Sex was determined by visual inspection of the gonads. Adults sampled 293 dph were used to take blood samples for the determination of the vitellogenin (VTG) content.
F1: On day 56 post-hatch, all surviving sh were sacrificed as above and measured for length and weight. Furthermore, samples were taken for the quantification of the VTG-content in whole body homogenates

VEHICLE CONTROL PERFORMED: yes

POST-HATCH DETAILS
- Begin of post-hatch period: When hatching commenced on day 4, the number of embryos hatched in each incubation cup was recorded. When hatching was > 90% complete on day 5 (0 dph) larvae in the incubation cups were reduced to 25 (24-25) organisms and released to each of the growth tanks (10 liters volume) without exposure to the air (by submerging the cups). The egg cups were removed. There were four replicates with a total of on average 99 larvae per concentration.
- Release of juveniles from incubation cups to test chamber: on day no. 56 dph juveniles were transferred to test tanks of 50 liters tank volume. Each tank contained an average of 48 (45-51) fish. The number of replicates was reduced to two. There were an average of 96 juveniles per concentration.

FERTILIZATION SUCCESS STUDY
- Number of eggs used: 45-50

Reference substance (positive control):

no

Key result

Duration:

0 d

Dose descriptor:

NOEC

Remarks:

days post hatching

Effect conc.:

0.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks on result:

other:

Remarks:

based on reduced hatching success in the F1 generation

Key result

Duration:

56 d

Dose descriptor:

NOEC

Remarks:

days post hatching

Effect conc.:

0.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other:

Remarks:

based on reduced survival in the F1 generation

Details on results:

The most sensitive endpoint over the period of the test was egg hatch and survival during the F1-generation. At the highest concentration of 0.040 mg/L, no individuals hatched at all. Consequently, all other endpoints measured at the end of exposure like growth, vitellogenin and survival were affected as there were no surviving fish at the highest concentration. Thus, the no observed effect concentration (NOEC) of the most sensitive endpoints was 0.020 mg/L. The lowest observed effect concentration (LOEC) was >0.020 mg/L. The results of the vitellogenin quantification suggested, that the test substance does not act as an estrogenic agonist in fathead minnow.

Reported statistics and error estimates:

Dunnett’s multiple means comparison test (Dunnett, 1955 & 1980) using mean values per replicate as experimental units was run. Bartlett’s Test (Bartlett, 1937) was used to check for the constancy of variance and Dunnett’s Test (Dunnett 1955, 1980) to provide the Lowest-Observed—Effect—Concentrations (LOEC)’s the No-Observed-Effect-Concentrations (NOEC)’s. Generally, the values of the treatments were compared to the pooled replicates of both the blank and vehicle control. If there was a significant difference between the values of the blank and the vehicle control, the values of the treatments were compared to those of the vehicle control only. The maximum acceptable toxicity concentration (MATC) was calculated as the geometric mean of the NOEC and the LOEC.

Table 1. Toxicity of the test substance to fathead minnow in a fish full life-cycle test

Biological Parameter			Effect concentration nominal (mg ai/L)
Generation	Days post hatch	Endpoint	NOEC	LOEC
F0	0 167-279 167-279 56 56 56 162 162 162 162 293 293 293	Egg hatchability Egg production %-Fertilization success Survival Length Weight Survival Length Weight GSI Survival Length Weight	0.040 0.040 0.040 0.040 0.040 0.040 0.040 0.040 0.040 0.040 0.040 0.040 0.040	>0.040 >0.040 >0.040 >0.040 >0.040 >0.040 >0.040 >0.040 >0.040 >0.040 >0.040 >0.040 >0.040
	293 293 293	GSI Vitellogenin –males Vitellogenin –females	0.040 0.040 0.040	>0.040 >0.040 >0.040
F1	0 56 56 56 56	Egg hatchability Survival Length Weight Vitellogenin	0.020 0.020 0.020 0.020 0.020	0.040 0.040 >0.020 >0.020 >0.020

 

 

Validity criteria fulfilled:

yes

Conclusions:

In a fish life cycle study performed in accordance with US EPA 72-5, the NOEC for fathead minnow exposed to the test substance was 0.02 mg/L based on reduced hatching success and survival in the F1 generation.

Executive summary:

The objective of this fish life cycle study performed according to US EPA 72 -5 and in compliance with GLP, was to assess the chronic toxicity of the test substance on the whole lifecycle (two generations) of fathead minnow (Pimephales promelas). Effects on hatchability, development, growth, reproduction and induction of vitellogenin (an oestrogen dependent process) were measured. Fish were exposed to nominal concentrations of 0.0025, 0.0050, 0.010, 0.020 and 0.040 mg/L, a dilution water and solvent control in a flow-through system. An average of 44 newly fertilised fathead minnow eggs were exposed in incubation cups. There were four replicates with an average of 174 eggs per concentration and controls. After hatching (number recorded), larvae numbers were impartially reduced to 25 and released into 15 litre glass aquaria (4 replicates with an average of 99 larvae per treatment). At 56 days post hatch juveniles were transferred to larger 50 litre aquaria (each an average of 48 fish) and the number of replicates was reduced to two. Subsequently, exposure during breeding was conducted (162 days post hatch till 293 days) with two replicate tanks each with 4 spawning chambers (16 fish per treatment). Spare fish were retained in order to supplement nonbreeding pairs. Resultant F1 eggs were treated as before in the F0 exposures until the juvenile stage. Biological observations were made in each test chamber on weekdays for abnormal behavioural and physical changes. On day 56-post hatch, all surviving fish were photographed to obtain lengths. F0 generation: on days 162 and 293 post-hatch, adults were sacrificed, measured and weighed. Gonads were dissected to determine the Gonadal Somatic Index (GSI). At 293 days blood samples were also taken for blood plasma vitellogenin determinations. F1 generation: at 56-days post-hatch, fish were sacrificed as above for length and weight measurement. Additional samples were taken for whole body homogenate vitellogenin determinations. Water samples for chemical analysis were taken at weekly intervals in alternate replicates. At the start and end of an exposure period (F0 and F1) samples were taken in all replicates. Samples were analysed by High Performance Liquid Chromatography (HPLC). Actual measured concentrations of the test substance were in close agreement with nominal values at both F0 (106-144% of nominal) and F1 (102-115% of nominal) generation exposure periods. Consequently, nominal values were used in endpoint calculations.

The lowest No-Observed-Effect Concentration (NOEC) for fathead minnow exposed to the test substance was 0.02 mg/L based on a statistically significant reduction in F1 egg hatch and survival in the 0.04 mg/L treatment in comparison with the control. Vitellogenin determinations indicate that the test substance does not act as an oestrogen agonist in fathead minnow.

Description of key information

All available data was assessed. The study representing the worst-case effects was included here and its effect value was used as the key value. Another study is included as supporting information.

Fish lifecycle toxicity study, fathead minnow, 293-d NOEC = 0.02 mg/L, reduction in F1 egg hatch and survival, US EPA 72 -5, Rufli 2003

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

0.02 mg/L

Additional information

There are two standard guideline and GLP-compliant studies available for this endpoint. The fish lifecycle toxicity study (Rufli 2003, US EPA 72 -5, Reliability 1) was selected as key study, because it represents the worst-case effects (i.e. showed lower NOEC value). The objective of the study was to assess the chronic toxicity of the test substance on the whole lifecycle (two generations) of fathead minnow (Pimephales promelas). Effects on hatchability, development, growth, reproduction and induction of vitellogenin (an oestrogen dependent process) were measured. Fish were exposed to nominal concentrations of 0.0025, 0.0050, 0.010, 0.020 and 0.040 mg/L, including a dilution water and solvent control in a flow-through system. An average of 44 newly fertilised fathead minnow eggs were exposed in incubation cups. There were four replicates with an average of 174 eggs per concentration and controls. After hatching (number recorded), larvae numbers were impartially reduced to 25 and released into 15 litre glass aquaria (4 replicates with an average of 99 larvae per treatment). At 56 days post hatch juveniles were transferred to larger 50 litre aquaria (each an average of 48 fish) and the number of replicates was reduced to two. Subsequently, exposure during breeding was conducted (162 days post hatch till 293 days) with two replicate tanks each with 4 spawning chambers (16 fish per treatment). Resultant F1 eggs were treated as before in the F0 exposures until the juvenile stage. Water samples for chemical analysis were taken at weekly intervals in alternate replicates. At the start and end of an exposure period (F0 and F1) samples were taken in all replicates. Samples were analysed by High Performance Liquid Chromatography (HPLC). Actual measured concentrations of the test substance were in close agreement with nominal values at both F0 (106-144% of nominal) and F1 (102-115% of nominal) generation exposure periods. Consequently, nominal values were used in endpoint calculations. The lowest No-Observed-Effect Concentration (NOEC) for fathead minnow exposed to the test substance was 0.02 mg/L based on a statistically significant reduction in F1 egg hatch and survival in the highest treatment (0.04 mg/L) in comparison with the control. At 0.04 mg/L, no individuals hatched at all. Consequently, all other endpoints measured at the end of the test were affected as there were no surviving F1 fish at the highest concentration. Vitellogenin determinations indicate that the test substance does not act as an oestrogen agonist in fathead minnow.

The supporting study was a juvenile growth study performed in rainbow trout following the OECD TG 204 and in compliance with GLP (Vial, 1992). The study carried out over 21 days. Ten fish were allocated per treatment and exposed to 0.0026, 0.0065, 0.016, 0.041, 0.10 mg test substance/L (actual test concentrations: 0.0020, 0.0043, 0.0090, 0.018, 0.069 mg test substance/L), including an untreated control and a vehicle blank. No mortality or visible adverse symptoms could be observed throughout exposure up to the highest treatment. The NOEC (21 days) for lethal effects was ≥ 0.069 mg/L. Statistical re-analysis of the data indicated that the NOEC (21 days) for non-lethal effects, weight and length was ≥ 0.069 mg/L.

Metabolites - available information

In addition to the studies with the test substance, one juvenile growth test is available with degradation product M6. As the dossier has been prepared to address the test substance itself, this study is not summarized as endpoint study record but is briefly discussed here. The exposure of Rainbow Trout to M6 over a period of 28 days resulted in an NOEC (28 days) with regard to both sublethal and lethal effects of 0.11 mg/L based on measured mean concentrations (Rufli, 1999).