Fenamiphos

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:

1988-10-20 to 1989-01-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

not specified

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling method: The control and the high, middle and low test concentrations were sampled and analyzed for the test substance concentrations once prior to the start of the definitive exposure. During the definitive test, samples were removed from all replicate test solutions and the controls on test day 0, 7, 14, 21, 28, 36, 42 and weekly thereafter. Each test solution sample was collected from the approximate midpoint of the exposure aquarium using a volumetric pipet in conjunction with a graduated cylinder. Three Quality Assurance (QA) blind samples were prepared at each sampling interval and remained with the set of exposure solution samples through extraction and analysis.

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: constant flow serial diluter
- Controls: negative control, solvent control
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium: 40 µL/L of acetone
- Test concentration separation factor: <= 2
- Evidence of undissolved material: no

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Source: Unfertilized rainbow trout eggs and sperm were individually packaged and shipped
under refrigeration from Mount Lassen Trout Farm

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: The eggs and sperm were received at 5°C. To fertilize the eggs, one half of the sperm was placed in a stainless steel bowl, the eggs were added, and then the bowl swirled to mix the contents thoroughly. The remaining sperm was added to the bowl and again mixed by swirling. A small amount of water removed from a control aquarium was then added to the sperm-egg mixture in the bowl, and the bowl was swirled again to ensure even distribution of the sperm over the eggs. The bowl containing the sperm and eggs was left undisturbed for approximately two minutes (long enough to complete the fertilization process), and the excess sperm was then rinsed from the eggs with control water. The embryos were allowed to remain undisturbed in control water during the water hardening period for approximately 1 hour. Upon completion of the water hardening period, fifty eggs were impartially selected and distributed to each of 28 egg incubation cups (50 eggs/cup).
- Subsequent handling of eggs: After all the eggs were counted, two egg cups were suspended in each duplicate test aquarium per exposure concentration and the control. Egg incubation cups were glass jars (5 cm O.D., 8 cm high) with 16-mesh Nitex screen bottoms. A rocker arm apparatus, as described by Mount (1968), was used to gently oscillate the incubation cups in the exposure solutions. Because of their extreme sensitivity to physical trauma during the early developmental stage, dead eggs within the egg cups were not removed between days 0 and 19 of the exposure period. After day 19, dead eggs, characterized by an opaque, white color, were removed and preserved in Stockard's solution (an 85 : 6 : 5 : 4 mixture of water, glycerine, formalin, and glacial acetic acid), which also served to clear the eggs, enabling a determination of embryonic development. A definitive determination of viability was made on day 19, when all the eggs exhibited wellpronounced embryonic development. Any egg exhibiting embryonic development (up to and including day 19), whether dead or alive, was considered fertile for purposes of determining percent viability. All non-viable eggs were discarded.


POST-HATCH FEEDING
- Start date: approx.. 11 days post-hatch
- Type/source of feed: larvae were fed live brine shrimp (Artemia salina) nauplii
- Frequency of feeding: three times daily on weekdays and at least twice daily on weekends and holidays; feeding was terminated 24 hours prior to test termination. Aquaria were brushed and siphoned when necessary to remove excess food and fecal matter.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

91 d

Post exposure observation period:

post exposre observation not performed

Hardness:

26 - 32 mg/L CaCO3

Test temperature:

11 °C

pH:

7.0 - 7.4

Dissolved oxygen:

9.6-9.7 mg/L

Salinity:

not applicable

Conductivity:

90 - 130 µmhos/cm

Nominal and measured concentrations:

Nominal: 1.1, 2.3, 4.5, 9.0 and 18 µg a.i./L; measured: 1.1, 2.1, 3.8, 7.4 and 15 µg a.i./L (mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: aquaria
- Type of flow-through: proportional diluter
- Renewal rate of test solutions: 6.5 aquarium volume replacements per day, increased to 10 aquarium volume replacements per day on day 19
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration: 2
- No. of vessels per control: 2
- No. of vessels per vehicle control: 2

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light, 8 hours darkness
- Light intensity: 26 - 60 footcandles

EFFECT PARAMETERS MEASURED: behavior and appearance of larvae observed daily, number of surviving larvae estimated twice weekly

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY: performed
- Results used to determine the conditions for the definitive study: yes

POST-HATCH DETAILS
- Begin of post-hatch period: 31

Reference substance (positive control):

not required

Duration:

91 d

Dose descriptor:

NOEC

Effect conc.:

>= 15 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

other: embryo viability

Duration:

91 d

Dose descriptor:

NOEC

Effect conc.:

>= 15 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks:

survival of hatch

Duration:

91 d

Dose descriptor:

NOEC

Effect conc.:

>= 15 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks:

larval survival (60 d post hatch)

Key result

Duration:

91 d

Dose descriptor:

NOEC

Effect conc.:

3.8 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

other: length and weight

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages: please refer to attached supplementary information
- Days to hatch or time to release of young: please refer to attached supplementary information
- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: please refer to attached supplementary information
- Observations on body length and weight of young and/or exposed parents at one or more time periods: please refer to attached supplementary information
- Number of healthy fish at end of test: please refer to attached supplementary information
- Type of and number with morphological abnormalities: please refer to attached supplementary information
- Type of and number with behavioural abnormalities: please refer to attached supplementary information
- Type and number of developmental effects: please refer to attached supplementary information
- Effect concentrations exceeding solubility of substance in test medium: no
- Incidents in the course of the test which might have influenced the results: no

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

All statistical conclusions were made at the 95% level of certainty except in the case of the
Chi-Square Goodness of Fit Test and the Bartlett's Test, in which the 99% level of certainty
was applied.

Please refer to attached supplementary information.

Validity criteria fulfilled:

yes

Conclusions:

In a study according to EPA-FIFRA Guideline 72-4 with rainbow trout (Oncorhynchus mykiss) the 91-day NOEC (based on length and weight) for the test item was determined to be 3.8 µg a.s./L (mean measured).

Executive summary:

The long-term toxicity of the test item to fish was evaluated in a 91-day flow-through Fish Early Life Stage Test according to EPA-FIFRA Guideline 72-4 using rainbow trout (Oncorhynchus mykiss) as test species. A constant flow serial diluter with a 50% dilution factor was used to prepare and deliver the selected test concentration range of 1.1, 2.3, 4.5, 9.0 and 18 µg a.i./L (nominal). The test item concentrations were analytically verified employing a HPLC-UV method to be 1.1, 2.1, 3.8, 7.4 and 15 µg a.i./L (mean measured). Egg incubation cups were glass jars (5 cm O.D., 8 cm high) with 16-mesh Nitex" screen bottoms. A rocker arm apparatus was used to gently oscillate the incubation cups in the exposure solutions. Because of their extreme sensitivity to physical trauma during the early developmental stage, dead eggs within the egg cups were not removed between days 0 and 19 of the exposure period. After day 19, dead eggs, characterized by an opaque, white color, were removed and preserved in Stockard's solution (an 85 : 6 : 5 : 4 mixture of water, glycerine, formalin, and glacial acetic acid), which also served to clear the eggs, enabling a determination of embryonic development. A definitive determination of viability was made on day 19, when all the eggs exhibited well- pronounced embryonic development. Any egg exhibiting embryonic development (up to and including day 19), whether dead or alive, was considered fertile for purposes of determining percent viability. All non-viable eggs were discarded. Percent viability was based on the number of fertile embryos produced from the initial number of eggs exposed. On test day 19, all live viable embryos from the two embryo incubation cups in each respective aquarium were counted and combined (e.g., 100 embryos per aquarium). Twenty live, viable embryos were then impartially selected and placed into an incubation cup which was suspended in the respective exposure aquarium. The remaining live, viable embryos in each aquarium were then suspended in a second incubation cup (e.g., 80 embryos per aquarium). The larvae which hatched from the twenty isolated embryos were used to initiate the swim-up stage of the study. Hatching was deemed complete (test day 31) when no more than 5 unhatched viable embryos remained in any embryo incubation cup. Calculations of percentage survival of organisms at hatch were based on the total number of live larvae per aquarium (i.e., total of the incubation cups per aquarium) after hatching was completed compared to the total number of viable embryos. To initiate the 60-day post-hatch larval exposure, the surviving larvae from each of the isolated embryo incubation cups within each replicate aquarium were released into their respective aquaria, providing a total of 20 larvae per replicate aquaria. Following 18 days of exposure each test aquarium was replaced weekly in order to minimize the increase in microbial activity. Preliminary investigations indicated that increased microbial activity was associated with the decrease in test material concentrations. The replacement of aquaria was conducted in an effort to maintain consistent exposure levels of the test material. Aquaria were replaced weekly, beginning on test day 19. The exchange of aquaria was not conducted prior to day 19 because the exposed embryos were sensitive to physical trauma during this early development stage. During the aquaria exchange, test organisms were temporarily placed in clean vessels containing solution removed from the respective exposure chambers. The old aquaria were removed and replaced with clean vessels. Test solutions and organisms were then returned to the exposure system within 3 - 4 minutes from the exchange was initiated. Old aquaria were cleaned for use during the next exchange. The exchange of aquaria was conducted on the day following analytical sampling. Organism handling during the transfer procedure was conducted using extreme care and caution. To minimize microbial activity in the exposure solutions, clean aquaria used during the vessel exchange were not equilibrated with the test material. When the rainbow trout reached the swim-up stage and began actively feeding (approximately 11 days post-hatch), larvae were fed live brine shrimp (Artemia salina) nauplii three times daily on weekdays and at least twice daily on weekends and holidays. Aquaria were brushed and siphoned when necessary to remove excess food and fecal matter. Behavior and appearance of larvae were observed daily and the number of surviving larvae was estimated twice weekly. At 60 days post-hatch the larvae from each aquarium were anesthetized with MS-222 (tricaine methanesulfonate) and percentage survival, mean total length, and mean wet weight were determined. The larvae were measured and weighed individually to calculate the mean and standard deviation of total length and wet weight. All statistical conclusions were made at the 95% level of certainty except in the case of the Chi-Square Goodness of Fit Test and the Bartlett's Test, in which the 99% level of certainty was applied. Exposure to all treatment levels of the test item assessed did not adversely affect embryo viability which ranged from 91 to 95 %. At the completion of the hatching period (test day 31), the survival of the rainbow trout embryos in all treatment levels ranged from 99 to 100 % and was comparable to the survival of the control embryos (pooled control data, 99 %). The incubation period of 31 days was generally consistent between treatment levels and the control, and was within the expected time interval for this species and the stated test temperature (11 ± 0.6 °C). By test day 47 (16 days post-hatch), nearly all exposed larvae in all treatment levels of the test item completed their development to the swim-up stage. At test termination (60 days post-hatch), mean survival of larvae exposed to all treatment-levels of the test item ranged from 88 to 100 % and was statistically comparable to control survival (pooled data, 96 %). Several fish at the highest treatment levels (15 µg a.i./L) were observed to exhibit a complete loss of equilibrium, were lethargic and had a darkened pigmentation. No significant toxicant-related sublethal effects were noted among fish exposed to the remaining concentrations of the test item (7.4 -1.1 µg a.i./L). Statistical comparison of the growth parameters indicated that larval length and larval weight were the most sensitive indicators of the toxicity of the test item. The mean total length of larvae exposed to 15 and 7.4 µg a.i./L was 56.3 and 57.2 mm, respectively, and was significantly (P < 0.05) less than the length of the control larvae (pooled data, 60.4 mm). Mean total length of larvae exposed to the remaining concentrations of the test item ranged from 58.4 to 59.7 mm and was not significantly different from the controls. Similarly, the mean wet weight of larvae exposed to 15 and 7.4  a.i./L was 1.7658 and 1.7671 grams, respectively, and was significantly (P <0.05) less than the weight of the control larvae (pooled data, 2.0393 g). Mean wet weight of larvae exposed to test item concentrations < 3.8 µg a.i./L ranged from 1.8156 to 2.0106 g and was unaffected as compared to the performance of control organisms. Based on the significantly (P < 0.05) reduced larval growth following 60 days post-hatch exposure to 7.4 µg a.i./L, the Lowest Observed Effect Concentration (LOEC) is estimated to be 7.4 µg a.i./L  (mean measured). The 91-day NOEC based on length and weight was determined to be 3.8 µg a.i./L (mean measured).

Description of key information

In a study according to EPA-FIFRA Guideline 72-4 with rainbow trout (Oncorhynchus mykiss) the 91-day NOEC (based on length and weight) for the test item was determined to be 3.8 µg a.s./L (mean measured).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

3.8 µg/L

Additional information

Experimental information on the chronic toxicity of the test substance to fish is available and summarized below.

 

The long-term toxicity of the test item to fish was evaluated in a 91-day flow-through Fish Early Life Stage Test according to EPA-FIFRA Guideline 72-4 using rainbow trout (Oncorhynchus mykiss) as test species. A constant flow serial diluter with a 50% dilution factor was used to prepare and deliver the selected test concentration range of 1.1, 2.3, 4.5, 9.0 and 18 µg a.i./L (nominal). The test item concentrations were analytically verified employing a HPLC-UV method to be 1.1, 2.1, 3.8, 7.4 and 15 µg a.i./L (mean measured). Egg incubation cups were glass jars (5 cm O.D., 8 cm high) with 16-mesh Nitex" screen bottoms. A rocker arm apparatus was used to gently oscillate the incubation cups in the exposure solutions. Because of their extreme sensitivity to physical trauma during the early developmental stage, dead eggs within the egg cups were not removed between days 0 and 19 of the exposure period. After day 19, dead eggs, characterized by an opaque, white color, were removed and preserved in Stockard's solution (an 85 : 6 : 5 : 4 mixture of water, glycerine, formalin, and glacial acetic acid), which also served to clear the eggs, enabling a determination of embryonic development. A definitive determination of viability was made on day 19, when all the eggs exhibited well- pronounced embryonic development. Any egg exhibiting embryonic development (up to and including day 19), whether dead or alive, was considered fertile for purposes of determining percent viability. All non-viable eggs were discarded. Percent viability was based on the number of fertile embryos produced from the initial number of eggs exposed. On test day 19, all live viable embryos from the two embryo incubation cups in each respective aquarium were counted and combined (e.g., 100 embryos per aquarium). Twenty live, viable embryos were then impartially selected and placed into an incubation cup which was suspended in the respective exposure aquarium. The remaining live, viable embryos in each aquarium were then suspended in a second incubation cup (e.g., 80 embryos per aquarium). The larvae which hatched from the twenty isolated embryos were used to initiate the swim-up stage of the study. Hatching was deemed complete (test day 31) when no more than 5 unhatched viable embryos remained in any embryo incubation cup. Calculations of percentage survival of organisms at hatch were based on the total number of live larvae per aquarium (i.e., total of the incubation cups per aquarium) after hatching was completed compared to the total number of viable embryos. To initiate the 60-day post-hatch larval exposure, the surviving larvae from each of the isolated embryo incubation cups within each replicate aquarium were released into their respective aquaria, providing a total of 20 larvae per replicate aquaria. Following 18 days of exposure each test aquarium was replaced weekly in order to minimize the increase in microbial activity. Preliminary investigations indicated that increased microbial activity was associated with the decrease in test material concentrations. The replacement of aquaria was conducted in an effort to maintain consistent exposure levels of the test material. Aquaria were replaced weekly, beginning on test day 19. The exchange of aquaria was not conducted prior to day 19 because the exposed embryos were sensitive to physical trauma during this early development stage. During the aquaria exchange, test organisms were temporarily placed in clean vessels containing solution removed from the respective exposure chambers. The old aquaria were removed and replaced with clean vessels. Test solutions and organisms were then returned to the exposure system within 3 - 4 minutes from the exchange was initiated. Old aquaria were cleaned for use during the next exchange. The exchange of aquaria was conducted on the day following analytical sampling. Organism handling during the transfer procedure was conducted using extreme care and caution. To minimize microbial activity in the exposure solutions, clean aquaria used during the vessel exchange were not equilibrated with the test material. When the rainbow trout reached the swim-up stage and began actively feeding (approximately 11 days post-hatch), larvae were fed live brine shrimp (Artemia salina) nauplii three times daily on weekdays and at least twice daily on weekends and holidays. Aquaria were brushed and siphoned when necessary to remove excess food and fecal matter. Behavior and appearance of larvae were observed daily and the number of surviving larvae was estimated twice weekly. At 60 days post-hatch the larvae from each aquarium were anesthetized with MS-222 (tricaine methanesulfonate) and percentage survival, mean total length, and mean wet weight were determined. The larvae were measured and weighed individually to calculate the mean and standard deviation of total length and wet weight. All statistical conclusions were made at the 95% level of certainty except in the case of the Chi-Square Goodness of Fit Test and the Bartlett's Test, in which the 99% level of certainty was applied. Exposure to all treatment levels of the test item assessed did not adversely affect embryo viability which ranged from 91 to 95 %. At the completion of the hatching period (test day 31), the survival of the rainbow trout embryos in all treatment levels ranged from 99 to 100 % and was comparable to the survival of the control embryos (pooled control data, 99 %). The incubation period of 31 days was generally consistent between treatment levels and the control, and was within the expected time interval for this species and the stated test temperature (11 ± 0.6 °C). By test day 47 (16 days post-hatch), nearly all exposed larvae in all treatment levels of the test item completed their development to the swim-up stage. At test termination (60 days post-hatch), mean survival of larvae exposed to all treatment-levels of the test item ranged from 88 to 100 % and was statistically comparable to control survival (pooled data, 96 %). Several fish at the highest treatment levels (15 µg a.i./L) were observed to exhibit a complete loss of equilibrium, were lethargic and had a darkened pigmentation. No significant toxicant-related sublethal effects were noted among fish exposed to the remaining concentrations of the test item (7.4 -1.1 µg a.i./L). Statistical comparison of the growth parameters indicated that larval length and larval weight were the most sensitive indicators of the toxicity of the test item. The mean total length of larvae exposed to 15 and 7.4 µg a.i./L was 56.3 and 57.2 mm, respectively, and was significantly (P < 0.05) less than the length of the control larvae (pooled data, 60.4 mm). Mean total length of larvae exposed to the remaining concentrations of the test item ranged from 58.4 to 59.7 mm and was not significantly different from the controls. Similarly, the mean wet weight of larvae exposed to 15 and 7.4  a.i./L was 1.7658 and 1.7671 grams, respectively, and was significantly (P <0.05) less than the weight of the control larvae (pooled data, 2.0393 g). Mean wet weight of larvae exposed to test item concentrations < 3.8 µg a.i./L ranged from 1.8156 to 2.0106 g and was unaffected as compared to the performance of control organisms. Based on the significantly (P < 0.05) reduced larval growth following 60 days post-hatch exposure to 7.4 µg a.i./L, the Lowest Observed Effect Concentration (LOEC) is estimated to be 7.4 µg a.i./L  (mean measured). The 91-day NOEC based on length and weight was determined to be 3.8 µg a.i./L (mean measured).