Methyl O-(4-amino-3,5-dichloro-6-fluoropyridin-2-yloxy)acetate

Long-term toxicity to fish

Administrative data

Endpoint:

fish, juvenile growth test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Nov- 23 Dec 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 215 (Fish, Juvenile Growth Test)

Version / remarks:

Draft version, 1992

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Methyl O-(4-amino-3,5-dichloro-6-fluoropyridin-2-yloxy)acetate

EC Number:

407-550-4

EC Name:

Methyl O-(4-amino-3,5-dichloro-6-fluoropyridin-2-yloxy)acetate

Cas Number:

69184-17-4

Molecular formula:

Hill formula: C8 H7 Cl2 F N2 O3 CAS formula: C8 H7 Cl2 F N2 O3

IUPAC Name:

methyl 2-[(4-amino-3,5-dichloro-6-fluoropyridin-2-yl)oxy]acetate

Details on test material:

- Name of test material (as cited in study report): Fluroxypyr methyl ester
- Physical state: solid, grey/black powder
- Analytical purity: 93.6%
- Lot/batch No.: HJ 11160402A
- Expiration date of the lot/batch: 18 November 1995
- Stability of test concentrations: verified by chemical analysis
- Storage condition of test material: in original container at 4°C

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Water samples (around 200 mL in duplicate) were taken from solvent control and each exposure level (samples from replicate vessels pooled) on days 0, 1, 7, 14, 21 and 28. The samples were acidified by the addition of 2 mL 1M HCl prior to analysing. One sample from each pair was used in the analysis, being the remaining sample stored at 4 °C
- Each sample bottle was shaken and the sample transferred to a measuring cylinder. Methanol (20 mL) was added to the bottle and the washings transferred to the measuring cylinder which was then diluted to 250 mL with dechlorinated water. Using an AutoTrace SPE Workstation, a sub-sample (100 mL) was pumped through a solid phase extraction cartridge (Varian Bond Elut C18, 3 cc) which had been previously conditioned with methanol (10 mL) and 0.01 M hydrochloric acid (10 mL). The cartridge was washed with 5 mL 0.01 M hydrochloric acid and dried with nitrogen (10 minutes). The retained fluroxypyr methyl ester and fluroxypyr were eluted with ethyl acetate (5.0 mL) into a test tube. The eluate was evaporated to dryness (Dri-block, 50°C under nitrogen). The extracts were redissolved in acetonitrile: water (50 : 50 v/v) and, following further dilution with the same solvent as required, an aliquot of the processed sample was analysed. Duplicate procedural recoveries and a blank were analysed concurrently with the samples

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was dissolved in a solvent to give a series of stock solutions of 0.1865, 0.597, 1.865, 5.97 and 18.65 mg/mL. These stock solutions were dispensed automatically by the dosing apparatus into the flowing test water to give the desired series of exposure levels
- Controls: control and solvent control
- Chemical name of vehicle: dimethylformamide
- Concentration of vehicle in test medium: 27 µL/L test medium

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Westacre Trout Farm, Norfolk, UK
- Weight at study initiation: 1.5-2.3 g (wet)
- Acclimation: the stock of fish was obtained from the supplier on 12 October 1993 and acclimatised to test conditions from 15 - 25 November
1993. Temperature remained at 14 °C and dissolved oxygen within the range 9.5 - 10.1 mgO2/L. The fish were fed daily to repletion with commercial trout pellets throughout the acclimatisation and was withheld for 24 hours prior to the performance of any fish weighing procedures. No medication was given during the acclimatisation period and mortalities were recorded as 0 % in the 7 days prior to the test.

FEEDING DURING TEST
- Food type: commercial trout pellets
- Amount: approximately 4% of bodyweight per day

Study design

Test type:

flow-through

Water media type:

freshwater

Total exposure duration:

28 d

Test conditions

Hardness:

166 ± 26 mgCaCO3/L

Test temperature:

13 ± 1 °C

pH:

7.7 ± 0.2

Dissolved oxygen:

9.6-9.7 mg O2/L

Nominal and measured concentrations:

Nominal: control, 0.005, 0.016, 0.050, 0.16, 0.5 mg/L
Measured: control, 0.0079, 0.015, 0.048, 0.18 and 0.58 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria (25 x 46 x 25 cm)
- Aeration: no supplementary aeration was provided
- Type of flow-through: peristaltic
- Renewal rate of test solution: solvent stock solutions were dispensed at the rate of 1.6085 mL/h into a diluent stream of 1000 mL/vessel/min
- No. of organisms per vessel: 16
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: initial loading of 1.5 g bw/L (static volume) and 0.02 g bw/L (volume in 24 h)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water filtered, dechlorinated and softened by passage through a water purification system. The water passes in sequence through a high pressure sand filter to remove colloids and particulate matter and a high grade activated carbon filter to remove chlorine and any organic contaminants. A proportion of the supply then passes through a water softener before final reverse osmosis treatment to produce a highly purified water supply. The two grades of dechlorinated water are then remixed in the ratio of approximately 1: 1 to give a supply with the desired water hardness


OTHER TEST CONDITIONS
- Photoperiod: 16 hours light/8 hours darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): growth (weight and length), mortality and sub-lethal effects at 0, 14 and 28 days

VEHICLE CONTROL PERFORMED: yes

Results and discussion

Effect concentrations

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.58 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth, mortality

Details on results:

- During the 28-day exposure period, no significant lethal or sub-lethal effects were evident in any test or control group. One fish was moribund from day 24 onwards at 0.58 mg/L and there was one mortality recorded ac 0.18 mg/L (nominal) on Day 15. Two fish from the solvent control group
and two from the 0.0079 mg/L group were lost between Days 14 - 15 when they jumped from their uncovered aquaria, but these incidents were not
test substance-related and did not affect the interpretation of the data. No other major adverse effeets were observed.
- Slight but significant differences between the growth rates of test and treated fish were apparent on occasions in none of these cases was there an
adverse effect attributable to treatment and no dose-response relationship was detected (the only effect observed was a slight inerease in growth rate)

Any other information on results incl. tables

Table 1. Mean bodyweight and standard length (± SD)

Mean measured concentration (mg/L)	Day 0		Day 14		Day 28
	Weight (g)	Length (cm)	Weight (g)	Length (cm)	Weight (g)	Length (cm)
Control   R1                R2	1.84(±0.19) 1.83(±0.24)	4.6(±0.2) 4.6(±0.2)	2.98(±0.50) 2.83(±0.58)	5.5(±0.3) 5.3(±0.4)	5.03(±0.88) 4.77(±1.13)	6.4(±0.4) 6.4(±0.5)
Solvent control    R1                R2                R3                R4	2.03(±0.18) 2.05(±0.21) 1.81(±0.25) 1.94(±0.15)	4.8(±0.2) 4.8(±0.3) 4.6(±0.3) 4.8(±0.2)	3.27(±0.48) 3.18(±0.44) 2.87(±0.52) 3.21(±0.40)	5.6(±0.4) 5.5(±0.3) 5.3(±0.4) 5.5(±0.3)	5.24(±0.88) 5.32(±0.80) 4.57(±0.84) 4.92(±0.74)	6.7(±0.4) 6.7(±0.4) 6.5(±0.5) 6.6(±0.5)
0.0079    R1                R2	1.85(±0.21) 1.92(±0.26)	4.7(±0.2) 4.8(±0.4)	2.84(±0.49) 2.97(±0.57)	5.3(±0.3) 5.4(±0.4)	4.30(±1.08) 4.68(±1.06)	6.4(±0.5) 6.6(±0.6)
0.015      R1                R2	1.85(±0.23) 1.83(±0.24)	4.8(±0.2) 4.8(±0.3)	3.28(±0.75) 3.19(±0.64)	5.7(±0.4) 5.5(±0.3)	4.84(±0.77) 5.49(±1.12)	6.7(±0.4) 6.9(±0.4)
0.048      R1                R2	1.87(±0.25) 1.84(±0.25)	4.7(±0.3) 4.8(±0.3)	3.17(±0.68) 3.17(±0.70)	5.6(±0.5) 5.7(±0.3)	5.76(±1.36) 5.36(±1.33)	7.0(±0.5) 7.0(±0.5)
0.18        R1                R2	1.86(±0.24) 1.94(±0.21)	4.8(±0.2) 4.9(±0.2)	3.08(±0.52) 3.20(±0.69)	5.5(-±0.5) 5.7(±0.4)	5.41(±1.38) 5.40(±1.04)	6.9(±0.5) 6.9(±0.4)
0.54        R1                R2	1.81(±0.26) 1.89(±0.22)	4.8(±0.3) 4.9(±0.3)	3.00(±0.51) 3.32(±0.78)	5.5(±0.5) 5.4(±0.4)	5.07(±1.24) 4.91(±1.14)	6.6(±0.5) 6.6(±0.5)

R1 Replicate 1

R2 Replicate 2

SD Standard deviation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes