2,3,5,6-tetrachloropyridine

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4th March 1991 to 10th December 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,3,5,6-tetrachloropyridine

EC Number:

219-283-9

EC Name:

2,3,5,6-tetrachloropyridine

Cas Number:

2402-79-1

Molecular formula:

C5HCl4N

IUPAC Name:

2,3,5,6-tetrachloropyridine

Details on test material:

Lot number WP-900914-748 of purity 98.6%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The concentration of 2,3,5,6¬tetrachloropyridine in the test vessels was analyzed on days 0, 2, and 4. On days 0 and 4, individual 8 mL samples were obtained from each of the 16 replicate test vessels. On day 2, composite samples were analyzed; four mL aliquots from each of 2 replicates (from each exposure concentration) were taken. Each sample was combined into a common 5-dram vial for analysis. The samples were extracted by adding 8 mL of cyclohexane and shaking for 30 minutes on a flatbed shaker. After centrifuging the samples for 5 minutes at 3000 RPM, the cyclohexane layer was transferred to autosampler vials for analysis by gas chromatography with electron capture detection (GC/ECD). Also, on days 0 and 4, the concentration of sym-tet in the mixing cell was analyzed to insure proper operation of the diluter system. Standards that covered the range of test concentrations were prepared on days 0, 2, and 4, and were analyzed along with the test vessel samples. The concentration of active ingredient on days 0 and 2 was calculated from response factor ratios and peak area responses, On day 4, the response of the ECD was non¬linear. This response was mathematically defined by generating a log¬transformed regression equation: Day 4 concentrations were determined by applying the regression equation to the sym-tet peak area responses. The concentration at each treatment level on day 4 was calculated using the following
log-transformed regression equation:

Y=e{(in (Z) X 1.0962)-7.9640]

Y=extract concentration (ng/mL), Z=SYM-TET peak area

Test solutions

Vehicle:

yes

Details on test solutions:

The nominal concentration of the test material stock solution was approximately 50 mg/mL a.i. (50.71 mg/mL). This was prepared by diluting 12.6775 g of the test material to 250 mL with DMF. Individual test solutions were prepared by the proportional flow-through diluter system described above. A DMF control was included because DMF was used as a vehicle for the test material. The nominal concentration of the vehicle control stock was 600 mg/mL. This was prepared by diluting 150 g of DMF to 250 mL with laboratory water. A 31.5% injection (of a 1 mL syringe) of the stock solution was delivered into the 2 L mixing cell to provide the vehicle control (vehicle control limit =0.1 mL carrier/L of test solution, DMF=0.9445 g/mL).

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Rainbow trout were obtained as eyed embryos from Mt. Lassen Trout Farms, Red Bluff, California on December 20, 1990. The embryos were hatched and reared in this laboratory at 12 ± 1 °C. After initial swim-up, the fish were held in a 430-L fiberglass tank at 12 ± 1°C. All fish were held on a 16-h light/8-h dark photoperiod and observed for at least 14 days prior to testing. During holding, they were fed a standard laboratory diet (Aquatic Diet No. 1, Harlan Sprague Dawley, Madison, Wisconsin) at least once daily. The test lot was acclimated to the test temperature and held without food for at least 48 hours before testing. Mortality of the test lot was less than 3 percent per day in the 5 days prior to testing. A daily record of conditions during the holding period was kept.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

Not applicable.

Test conditions

Hardness:

Control High Dose
Hardness (as CaCO3) 72 mg/L 72 mg/L

Test temperature:

12.5± 0.5°C

pH:

Control High Dose
pH 7.9 7.9

Dissolved oxygen:

Dissolved Oxygen (mg/L)
(0-48h) 9.4 - 10.4 (> 87% of air saturation)
(72-96h) 9.4 - 10.2 (> 87% of air saturation)

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal Concentration (mg/L) Analysed Concentration (mg/L.)
Oh 48h* 96h Mean** % Nominal
Water Control A BLQa BLQ BLQ NA NAb
Water Control B BLQ - BLQ - -

DMFControl A BLQ BLQ BLQ NA NA
DMF Control B BLQ - BLQ

0.19 A 0.18 0.24 0.17 0.20 105.3
0.19 B 0.17 - 0.17 - -

0.32 A 0.34 0.34 0.33 0.33 103.1
0.32 B 0.31 - 0.33 - -

0.54 A 0.64 0.55 0.59 0.56 103.7
0.54 B 0.51 - 0.49 - -

0.90A 1.02 0.98 0.88 0.94 104.4
0.90B 0.93 - 0.86 - -

1.5 A 2.24 1.64 1.73 1,72 114.7
1.5 B 1.63 - 1.42 - -

2.5 A 3.92 3.30 3.30 3.09 123.6
2.5 B 2.50 - 2.21 - -
* The 48h samples were composite samples.
** Mean Concentration= Sum of Daily Averages/3
a BLQ=Below Limit of Quantitation (4 ng/mL).
b NA=Not Applicable.

Details on test conditions:

Laboratory Water.
The raw water was from the upper Saginaw Bay of Lake Huron off Whitestone Point and was limed and flocculated with ferric chloride by the City of Midland to reduce its hardness. Prior to its use in the laboratory, the water was sand-filtered, pH-adjusted with CO2, carbon-filtered, and UV
irradiated. Laboratory water was also analyzed weekly for pH, conductivity, alkalinity, and hardness; quarterly for selected inorganics.

Test Organisms.
Rainbow trout were obtained as eyed embryos from Mt. Lassen Trout Farms, Red Bluff, California on December 20, 1990. The embryos were hatched and reared in this laboratory at 12 ± 1 °C. After initial swim-up, the fish were held in a 430-L fiberglass tank at 12 ± 1°C. All fish were held on a 16-h light/8-h dark photoperiod and observed for at least 14 days prior to testing. During holding, they were fed a standard laboratory diet (Aquatic Diet No. 1, Harlan Sprague Dawley, Madison, Wisconsin) at least once daily. The test lot was acclimated to the test temperature and held without food for at least 48 hours before testing. Mortality of the test lot was less than 3 percent per day in the 5 days prior to testing. A daily record of conditions during the holding period was kept.

Test Substance Delivery System.
An intermittent-flow proportional diluter was used for dosing of the test vessels. This system is designed to. deliver up to six test concentrations, a vehicle control, and a laboratory water control. The diluter was calibrated so that the concentration of test material, in each treatment below the high concentration, was approximately 60% of that in the next higher dose level. The diluter operates as follows: a precision dosing system (MicroMedic Automatic Pipette System) delivers a specific amount of the test material from a concentrated stock solution to the mixing chamber (-5 L) where it is mixed with water. The mixing chamber has a recirculating pump that thoroughly mixes the stock solution and water. After approximately four minutes of mixing, the test solution is distributed to the appropriate toxicant cells. When the diluter cycles, the test solution from each toxicant cell is mixed with water from its respective water cell and flows into splitting chambers. Silicone delivery tubes from these chambers provide each replicate test vessel with the test solution (--1L per aquaria). The location of the test vessels within the water trough was randomized using a table of random digits [9]. The diluter was calibrated prior to the beginning of the test. During the test, the diluter was set to provide at least six volume turnovers in each vessel every 24h. General operation of the diluter was checked daily using a checklist.

Test Vessels.
Testing aquaria are constructed of double-strength glass and clear silicone adhesive; the test vessels measure 30 X 15 X 14 cm. The aquaria were fitted with glass covers to retard evaporation and to prevent the test organisms from escaping. A.volume of -4L per vessel was maintained by Nitex®R, screen¬covered drain guards. Each test vessel was uniquely labeled for identification purposes. These test vessels were held in a circulating water bath with the temperature controller set to maintain 12.5 ± 0.5°C. During the test, a 16-h light/8-h dark photoperiod was provided using cool-white fluorescent lighting on an automatic timer.

Test Procedures.
Preliminary testing (2/8 - 12/91) indicated that the 96-h LC50 was less than 5 mg/L a.i. (nominal). Based on this data and the results of the range-finding probe (3/4/91 - 3/8/91), the definitive test (6/21/91 - 6/25/91) was set using six nominal treatment levels (0.19, 0.32, 0.54, 0.9, 1.5, and 2.5 mg sym¬tet/L), a DMF (N,N-dimethylformamide) control (Fisher Scientific, Lot #902364), and a laboratory water control. Groups of ten fish were exposed at each treatment level and control (5 fish/replicate; 2 replicates/concentration). Fish were impartially added to the test vessels and were not fed during the test. The nominal concentration of the test material stock solution was approximately 50 mg/mL a.i. (50.71 mg/mL). This was prepared by diluting 12.6775 g of the test material to 250 mL with DMF. Individual test solutions were prepared by the proportional flow-through diluter system described above. A DMF control was included because DMF was used as a vehicle for the test material. The nominal concentration of the vehicle control stock was 600 mg/mL. This was prepared by diluting 150 g of DMF to 250 mL with laboratory water. A 31.5% injection (of a 1 mL syringe) of the stock solution was delivered into the 2 L mixing cell to provide the vehicle control (vehicle control limit =0.1 mL carrier/L of test solution, aDMF=0.9445 g/mL).

Data Recording.
The test vessels were observed daily for mortality (no response to gentle prodding and no opercular movement) and sublethal effects. Dead fish were removed at this time and a record of mortality and sublethal effects was maintained. Any sublethal effects were noted and recorded, e.g. swimming at the surface, complete or partial loss of body equilibrium, lethargy, erratic movement, and exophthalmia.

Measurement of dissolved oxygen, pH, and temperature was made at initiation of testing and daily thereafter in all test and control vessels with surviving fish. Temperature was also recorded continuously in one representative vessel. Weight and standard length measurements were made on the laboratory water control and vehicle control fish at test termination. Fish were euthanatized using tricaine methanesulfonate (Crescent Research Chemicals, Lot No. 2876G1) prior to measurement.

Laboratory Lighting
Light Intensity (lux) 1364

Test Substance Water Quality, Control High Dose
Hardness (as CaCO3) 72 mg/L 72 tng/L
Alkalinity (as CaCO3) 43 mg/L 42 rng/L
Conductivity (umho/cm) 145 150
pH 7.9 7.9

Test Vessels
Temperature Range (°C) 12.1 - 12.8
pH Range 7.7- 8.0
Dissolved Oxygen (mg/L)
(0-48h) 9.4- 10.4 (> 87%a of air saturation)
(72-96h) 9.4- 10.2 (> 87% of air saturation)

Test Organisms
Lot Number RT12209OLR
Mean Standard Length (Range) 35.4 mm (32 - 39 mm)
Mean Weight 0.60 g
Loading 0.05 g fish/L of test solution/day

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Evaluation of the Toxicity of Sym-tet to the Rainbow Trout: Concentration-Response Data.

Mean Analyzed Number Dead
Concentration (mg/L) N 24h 48h 72h 96h
Water Control A 5 0 0 0 0
B 5 0 0 0 0
DMF Control A 5 0 0 0 0
B 5 0 0 0 0
0.20 A 5 0 0 0 0
B 5 0 0 0 0
033 A 5 0 0 0 0
B 5 0 0 0 0
0.56 A 5 0 0 0 0a
B 5 0 0 0 0a
0.94 A 5 0a 0a 0a 0a
B 5 0 0 0a 0a
1.7 A 5 0a 3a 3a 3a
B 5 Oa 4a 4a 4a
3.1 A 5 5 5 5 5
B 5 5 5 5 5



LC50 (mg/L)b 2.3 1.5 1.5 1.5
95% Confidence Interval (mg/L)c 1.7-3.1 0.94-3.1 0.94-3.1 0.94-3.1

a Surviving fish exhibited loss of equilibrium, lethargy, and erratic body movements.
b LC50 by non-linear interpolation.
c 95% CI by binomial method.

Results with reference substance (positive control):

See above.

Reported statistics and error estimates:

Statistical Analysis.
A computer program was used to calculate the LC50 values and corresponding 95% confidence intervals. Mean analyzed concentrations at test initiation were used to calculate LC50 values. The program has three methods available: probit analysis, moving average angle analysis, and binomial probability. The probit analysis and moving average methods calculate both the estimated LC50 value and its confidence interval. The binomial method calculates only the confidence interval, while a point estimate of the LC50 value is obtained using a non-linear interpolation, i.e., log transformation of the concentration and angle transformation of the number dead.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Statistical analysis of the data yielded a 96-h LC50 of 1.5 mg/L a.i. with a 95% confidence interval of 0.94 - 3.1 mg/L. The 96-hour mortality threshold concentration was 0.94 mg/L. Sublethal effects, such as lethargy, surface swimming, and loss of equilibrium were not noted below the 0.56 mg/L dose level. Therefore, the no-observed-effect concentration (NOEC) was 0.33 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The LC50 for sym-tet was determined to be 1.5 mg/L with a 95% confidence interval of 0.94 - 3.1 mg/L. The 96-hour mortality threshold concentration was 0.94 rng/L and the no-observed-effect concentration (NOEC) was 0.33 mg/L.

Executive summary:

The study was conducted in accordance with OECD Guideline Number 203 and was designed as a 96 -hour flow-through test with analyzed exposure concentrations. Groups of ten rainbow trout were exposed to mean analyzedtreatment levels of 0.20, 0.33, 0.56, 0.94, 1.7, and 3.1 mg/L active ingredient (a.i.), aDMF (N,N-dimethylformamide) control, and a laboratory water control.

The LC50 for sym-tet was determined to be 1.5 mg/L with a 95% confidence interval of 0.94 - 3.1 mg/L. The 96-hour mortality threshold concentration was 0.94 rng/L and the no-observed-effect concentration (NOEC) was 0.33 mg/L.