pymetrozine (ISO); (E)-4,5-dihydro-6-methyl-4-(3-pyridylmethyleneamino)-1,2,4-triazin-3(2H)-one

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:

29 Jun 1994 to 27 Sep 1994

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:

Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) 72-4, 1993

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Analytical determination of teat material concentration (active ingredient) was performed with samples collected from each replicate test vessel at test initiation, every 7 days after test initiation, and at test termination. The samples were stored in either 40 ml amber glass vials or 120 mL amber glass bottles. Samples or extracts were refrigerated if not analyzed on the day of collection. samples collected from replicate 1 test vessels on day 0 and every 14 days thereafter, and samples collected from the second replicate on day 7 and every 14 days thereafter were analyzed. The alternate replicate samples were refrigerated for possible later analysis. Recoveries for several of the primary samples analyzed were below expected values. It appears that these low recoveries were the result of eluting the samples too quickly through the BOND-ELUT® columns. The back-up samples were subsequently analyzed and the resulting values were closer to the expected values. The results of both initial and backup analyses are reported.

Vehicle:

yes

Remarks:

dimethylformamide

Details on test solutions:

- Method: Stock solutions were prepared at 13,000 mg/L by either combining 13.2 g of test substance and dimethylformamide to a total volume of 1000 mL, or 39.6 g of test substance and dimethylformamide to a total volume of 3000 mL. The test substance was supplied with an intermittent flow proportional diluter that maintained the final test concentrations during the study.
- Solvent: The concentration of dimethylformamide was maintained under 2 mL/L to reduce the possible effect of solvent on the embryos and to attempt to eliminate the need for aeration caused by the buildup of a biological slime layer in the test vessels.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Age at study initiation: embryos, approximately 3.5 hours old at test initiation.
- Embryo health at study initiation: The embryos were free of apparent sickness, injuries, and abnormalities at the beginning of the test.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Subsequent handling of eggs: The eggs and milt were combined in dilution water adjusted to 10.8°C and gently mixed. Excess sperm and debris were rinsed away from the embryos after a 1 minute mixing period and embryos were water hardened for approximately 3.5 hours in chilled, flowing dilution water. Embryos were transferred with a wide bore pipette. The identification of the test organisms was verified at the conclusion of the test.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

90 d

Remarks on exposure duration:

30 days hatching period; 60 days post-hatch exposure

Hardness:

40 - 48 mg/L CaCO3

Test temperature:

10.8 - 13.0 °C

pH:

7.2 - 7.7

Dissolved oxygen:

8.6 - 11.4 mg/L

Salinity:

Not reported

Conductivity:

160 - 220 μmhos/cm

Nominal and measured concentrations:

- Nominal concentrations: 0.0 (blank control), 0.0 (solvent control), 0.78, 1.6, 3.3, 6.5, and 13 mg/L
- Mean measured concentrations: ND (blank control), ND, (solvent control), 0.555, 1.27, 2.82, 5.64 and 11.7 mg/L A detailed overview of the measured concentrations over time is presented in 'Any other information on materials and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
- Emybro cups: Embryos were exposed in cages that consisted of glass cylinders that were closed at one end with Nitex(c) screen. The cages were oscillated slowly to assure an adequate flow of media around the embryos. Hatching was at least 95% complete in the control exposure after 30 days of exposure.
- Test vessel: The test was performed in 20L glass aquaria (approximately 20 cm in width, 40 cm in length, and 25 cm in height) that contained 15 liters of test solution (water depth was approximately 18 cm).
- Aeration: Aeration was not required to maintain the dissolved oxygen concentration above acceptable levels.
- Type of flow-through: The test substance was supplied to the test vessels under flowthrough conditions by an intermittent flow proportional diluter. The diluter allowed test media to contact only glass or Teflon® surfaces. The diluter was calibrated before and after the test. During the test the diluter was activated 16,347 times, resulting in an average of 6.1 volume additions per 24 hours in each test vessel.
- No. of fertilized eggs/embryos per embryo cup: 40; two cages, each containing 20 embryos, were suspended in each replicate test vessel.
- No of organisms per vessel: 15
- No. of vessels per concentration: 2
- No. of vessels per control: 2
- No. of vessels per vehicle control: 2
- Control data: Ninety three percent of the control and solvent control embryos hatched, and 93 to 100% of the fish in the control and solvent control replicates survived the 60-day post hatch exposure. Control and solvent control fish had an average wet weight (blotted) of 1.08 g and an average total length of 46 mm at the end of the test. The relative standard deviation of the weights of surviving fish in the control and solvent control test chambers was less than 40%.
- Biomass loading rate: Maximum loading rate during the toxicity test was approximately 1.08 g/L at any time and approximately 0.18 g/L/24 hours.
- Feeding: During the first 7 days following swim up fish were fed commercial dry starter chow and live, newly hatched Artemia salina nauplii ad libitum 3 times per day. After 7 days they were supplied with commercial dry starter chow and/or live, newly hatched Artemia salina nauplii at least two times each day except during the final 41 hours of the test. Exposure vessels were cleaned daily after the initiation of feeding.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water used for acclimation of test organisms and for all toxicity testing was dechlorinated tapwater. The dilution water was adjusted to a hardness of 40 to 48 mg/L as CaCO3 and stored in 500-gallon polyethylene tanks where it was aerated and repeatedly passed through a particle filter, ultraviolet sterilizer, and activated carbon.
- Alkalinity: 21 - 30 mg/L as CaCO3

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Embryos were initially maintained in darkness except for a brief period each day when survival and water quality data were collected. Following hatch, a 16 hour light and 8 hour dark photoperiod was automatically maintained with a 15 minute transition period was provided between dark and light.
- Light quality: Cool-white fluorescent lights
- Light intensity: Approximately 35 footcandles to each test vessel

WATER QUALITY MEASUREMENTS
Dissolved oxygen (YSI Model 57 meter), pH (Beckman model pH1 12 meter), conductivity (Cole Parmer meter), and temperature (Beckman model pH1 12 meter) were measured and recorded at the beginning of the test and daily thereafter in each test chamber as long as living animals were present in those vessels. The temperature in one vessel was continuously recorded. Hardness and alkalinity were measured in the dilution water weekly during the test.

EFFECT PARAMETERS MEASURED: Survival, sub-lethal effects, length and weight
The number of surviving organisms and the occurrence of sublethal effects (loss of equilibrium, erratic swimming, loss of reflex, excitability, discoloration, or change in appearance or behavior) were determined visually and recorded initially and at 24 hour intervals. Dead test organisms were removed when first observed. Fish and a ruled measuring device were photographed in the test vessels with the water depth reduced to a minimum at 31 days post hatch and the photographs were placed in the study file. At the conclusion of the test, surviving fish were blotted on paper towels and immediately weighed and measured.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
A screening test was conducted under static conditions with newly fertilized rainbow trout embryos. Nominal concentrations were 0.003, 0.03, 0.3, 3, and 30 mg/L with a maximum solvent concentration of 2 mL/L dimethylformamide. The test media was renewed twice during the test. After 29 days of exposure there was greater than 50% survival at 0.003, 0,03, 0.3, and 3 mg/L, and 10% survival at 30 mg/L test substance.

Reference substance (positive control):

no

Key result

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

11.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: sublethal effects

Remarks on result:

other: See 'Any other information on results' for an overview of all derived effect values.

Details on results:

A complete overview of the results is presented in 'Any other information on results incl. tables'.
- Test substance solubility: Insoluble material was not observed in any test vessel during the study.
- Control hatch success: 93% of the control and solvent control embryos hatched, and 93 to 100% of the fish in the control and solvent control replicates survived the 60-day post hatch exposure.
- Survival: At the termination of the test there was at least 86.7% survival in the control, solvent control, and at all tested concentrations (survival was not significantly different from the control at any concentration).
- Sublethal effects: The percent of healthy embryos after 48 hours, the time to first hatch (day 28), time to swim up and first feeding (day 41), and mortality and sublethal effects of embryos, larvae, and juveniles 7, 14, 21 days post hatch were identical for the controls and all treatments. No sublethal effects were observed in any test vessel containing the test substance during the test.
- Length and weight: The mean total length and mean wet weight of surviving fish exposed to the test substance were not significantly different than the controls at any concentration.

Reported statistics and error estimates:

See 'Any other information on materials and methods incl. tables' for a description of the statistical methods used.

Table: Survival of Rainbow trout, Oncorhynchus mykiss, at 48 hours, at hatch, and at 7, 14 , 21, 30 and 60 days post hatch, during the toxicity test with the test substance(1)

Mean measured concentration of the test substance (mg/L)	Rep.(2)	Percent of survival at 48 hours	Percent survival at hatch	Percent survival after hatch and thinning (days post hatch)
			Day 31	7	14	21	30	60
ND (control)(3)	1	100.0	92.5	100.0	100.0	100.0	100.0	100.0
	2	100.0	92.5	100.0	100.0	100.0	100.0	93.3
	Mean	100.0	92.5	100.0	100.0	100.0	100.0	96.7
ND (solvent control)	1	100.0	95.0	100.0	100.0	100.0	100.0	93.3
	2	100.0	90.0	100.0	100.0	100.0	100.0	93.3
	Mean	100.0	92.5	100.0	100.0	100.0	100.0	93.3
0.555	1	100.0	95.0	100.0	100.0	100.0	100.0	100.0
	2	100.0	90.0	100.0	100.0	100.0	93.3	93.3
	Mean	100.0	92.5	100.0	100.0	100.0	96.7	96.7
1.27	1	100.0	97.5	100.0	100.0	100.0	100.0	100.0
	2	100.0	97.5	100.0	100.0	100.0	100.0	93.3
	Mean	100.0	97.5	100.0	100.0	100.0	100.0	96.7
2.82	1	100.0	97.5	100.0	100.0	100.0	100.0	93.3
	2	100.0	97.5	100.0	100.0	100.0	100.0	100.0
	Mean	100.0	97.5	100.0	100.0	100.0	100.0	96.7
5.64	1	100.0	100.0	100.0	100.0	100.0	100.0	100.0
	2	100.0	92.5	100.0	100.0	100.0	93.3	93.3
	Mean	100.0	96.3	100.0	100.0	100.0	96.7	96.7
11.7	1	100.0	92.5	100.0	100.0	100.0	100.0	100.0
	2	100.0	90.0	100.0	100.0	100.0	100.0	86.7
	Mean	100.0	91.3	100.0	100.0	100.0	100.0	93.4

1) No sublethal effects were noted at any time during the test.

2) Rep. = replicate.

3) ND = none detected at or above the analytical detection limit (0.100 mg/L).

 

Table: Length and weight of rainbow trout, Oncorhynchus mykiss, at the conclusion of the toxicity test with the test substance.

Mean measured concentration of the test substance (mg/L)	Replicate	Total length (mm)	Wet weight (g)
		mean ± std. dev(1)	mean ± std. dev
ND control (2)	1	45.7 ± 1.9	1.07 ± 0.2
	2	43.8 ± 2.2	1.13 ± 0.2
	Total	44.8 ± 2.3	1.10 ± 0.2
ND (solvent control)	1	46.0 ± 2.5	1.09 ± 0.1
	2	45.8 ± 2.8	1.04 ± 0.2
	Total	45.9 ± 2.7	1.06 ± 0.2
0.555	1	46.9 ± 1.6	1.08 ± 0.1
	2	47.1 ± 1.8	1.17 ± 0.1
	Total	47.0 ± 1.7	1.12 ± 0.1
1.27	1	46.9 ± 1.8	1.12 ± 0.1
	2	47.2 ± 2.0	1.11 ± 0.1
	Total	47.0 ± 1.9	1.12 ± 0.1
2.82	1	46.0 ± 1.7	1.04 ± 0.2
	2	47.1 ± 1.8	1.06 ± 0.2
	Total	46.6 ± 1.9	1.05 ± 0.2
5.64	1	45.9 ± 2.3	1.14 ± 0.2
	2	47.6 ± 2.3	1.12 ± 0.2
	Total	46.7 ± 2.3	1.13 ± 0.2
11.7	1	46.1 ± 1.9	1.10 ± 0.1
	2	45.9 ± 1.8	1.07 ± 0.1
	Total	46.0 ± 1.8	1.09 ± 0.1

(1) std. dev. = standard deviation

(2) ND = none detected at or above the analytical detection limit (0.100 mg /L).

 

Table: No observed effect levels (NOELS), lowest observed effect levels (LOELs) , and maximum acceptable toxic ant concentrations (MATCs) from the toxicity test with rainbow trout, Oncorhynchus mykiss, and the test substance.

Biological endpoint	NOEL	LOEL	MATC
Normal embryos at 48 hours	11.7 mg/L	>11.7 mg/L	>11.7 mg/L
Normal embryos at hatch	11.7 mg/L	>11.7 mg/L	>11.7 mg/L
Time to hatch	11.7 mg/L	>11.7 mg/L	>11.7 mg/L
Time to swim up and first feeding	11.7 mg/L	>11.7 mg/L	>11.7 mg/L
Surviving fish:
7 days after hatch	11.7 mg/L	>11.7 mg/L	>11.7 mg/L
14 days after hatch	11.7 mg/L	>11.7 mg/L	>11.7 mg/L
21 days after hatch	11.7 mg/L	>11.7 mg/L	>11.7 mg/L
30 days after hatch	11.7 mg/L	>11.7 mg/L	>11.7 mg/L
60 days after hatch	11.7 mg/L	>11.7 mg/L	>11.7 mg/L
Healthy fish:
7 days after hatch	11.7 mg/L	>11.7 mg/L	>11.7 mg/L
14 days after hatch	11.7 mg/L	>11.7 mg/L	>11.7 mg/L
21 days after hatch	11.7 mg/L	>11.7 mg/L	>11.7 mg/L
30 days after hatch	11.7 mg/L	>11.7 mg/L	>11.7 mg/L
60 days after hatch	11.7 mg/L	>11.7 mg/L	>11.7 mg/L
Length	11.7 mg/L	>11.7 mg/L	>11.7 mg/L
Wet Weight	11.7 mg/L	>11.7 mg/L	>11.7 mg/L

Validity criteria fulfilled:

yes

Remarks:

see 'Any other information on materials and methods incl. tables'

Conclusions:

No effects were observed in any of the treatments. The 60-day NOEC was 11.7 mg/L.

Executive summary:

The early life stage toxicity of the test substance to the rainbow trout, Oncorhynchus mykiss, was determined in a study according to FIFRA Guideline 72-4(a) and in compliance with GLP criteria. The test was conducted for 90 days. The test was performed under flow-through conditions with five concentrations of test substance, a solvent control (1.0 ml/L dimethylformamide), and a dilution water control at 12 ± 1.5°C. The dilution water was dechlorinated tap water and adjusted to a hardness of 40 to 48 mg/L as CaCO3. Nominal concentrations of the active ingredient were: 0.0 mg/L (control and solvent control), 0.78, 1.6, 3.3, 6.5, and 13 mg/L test substance. The highest nominal concentration tested was 13 mg/L in order to maintain the concentration of dimethylformamide under 2 mL/L. This was done to reduce the possible effect of solvent on the embryos and to attempt to eliminate the need for aeration caused by the buildup of a biological slime layer in the test vessels. Mean measured concentrations of test substance were ND (not detected at or above the detection limit of 0.100 mg/L; control and solvent control), 0.555, 1.27, 2.82, 5.64, and 11.7 mg/L. Fertilized eggs were approximately 3.5 hours post-fertilization at the beginning of the study. Measured biological parameters were the survival of first generation embryos and fish, the time to hatch, swim up, and feeding, the length and weight of the surviving fish, and sublethal effects. Survival in the control, solvent control, and all treatments was at least 90% at hatch (day 30) and at least 86.7% 60 days after hatch. No sublethal effects were noted in any test vessel during the test, and the time to hatch, swim up and feeding (day 41) were identical for the controls and all treatments. The mean total length and wet weight of surviving fish exposed to the test substance were not significantly different from the controls at any tested concentration. Based on these findings, the 60-d NOEC was determined to be 11.7 mg/L.

Description of key information

All available data was assessed and the studies representing the worst-case effects were included as key studies. Other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA.

- 60-d NOEC = 11.7 mg/L in freshwater fish (Oncorhynchus mykiss), EPA OPP 72-4(a), Boeri, 1995

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

11.7 mg/L

Additional information

Table: Overview of available data on the long-term toxicity to fish

Species	Guideline / GLP	Endpoint	Effect value	Comment	Reference
Oncorhynchus mykiss(freshwater)	FIFRA 72-4 / GLP	60-d NOEC	11.7 mg/L	Flow-through regime. Mean measured concentrations were used for the effect values. No effects were observed in any of the treatments.	Boeri, 1995