2,3,5,6-tetrachloropyridine

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 March 1991 to 7 January 1992

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:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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:

Reference: AGR 289294
Purity = 98.6%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Chemical Analysis. A set of standards was prepared with sym-tet in order to quantify the amount of test material in the test solutions. On day 0 unfiltered samples were analyzed on each replicate test concentration and controls. Because of the evidence of undissolved test material in the test beakers and the large difference in results between replicates [average relative standard deviation (RSD) ranged from 21.6 to 116%] in the exposure concentrations on the day 0 analyses, filtered samples were also included on the day 2 analyses.

The filtered samples were prepared by taking 10 mL aliquots from each test beaker and filtering through a 10 mL disposable polypropylene syringe fitted with an Alltech Anotop 25-plus syringe filter (0.20 mm) into 5-dram vials. Both types of samples, the filtered and unfiltered, were then extracted with an equal volume of cyclohexane and shaken for 30 minutes on a flatbed shaker. After shaking, the samples were centrifuged for approximately one minute at 3000 RPM. The cyclohexane extracts were transferred to uniquely numbered auto-sampler vials and analyzed using capillary gas chromatography with electron capture detection (CpGC/ECD).

The amount of sym-tet in the extract was determined using the log-transformed regression equation and the calculated average peak response from duplicate injections of each test sample.

Test solutions

Vehicle:

yes

Details on test solutions:

Due to difficulty in dissolving the test material in aqueous solution, a solvent, dimethyl formamide (DMF), was used as a carrier. An initial stock solution of 301 mg/mL (15.05g of test material/50 mL DMF) was prepared in order that the maximum solvent limit of 0.1 mL DMF/L test solution would not be exceeded in setting the highest test concentration. The definitive test was set with six nominal concentrations, 30, 18, 10.8, 6.48, 3.89, 2.33 mg/L, a DMF control (N,N¬dimethyl formamide, Fisher Scientific, Lot #902364), and a laboratory water control and was conducted under flow-through conditions. Thirty instars were exposed to each test concentration; two daphnids/tube, five tubes/replicate, three replicates/concentration. The instars were not fed during the test. An intermittent-flow proportional diluter was used for dosing the test vessels.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Instars (i.e. daphnids less than 24-h old) from a laboratory-reared culture of Daphnia magna were used to initiate the test. Rearing conditions wereas follows:
illumination (cool-white fluorescent) 2500 ± 110 lux; 16-h light/8-h dark photoperiod;
temperature 20 ± 1 °C. Daphnids were fed an algal diet of Selenastrum capricornutum three times weekly.
The day before instars were needed for testing, stock tanks with daphnids greater than 14 days old were removed from the incubator and the instars separated from adults by gently lifting the screened insert from the 2 L stock tank and releasing instars through the nylon mesh screen while retaining the adult daphnids. The screened insert with adult daphnids was then placed in another stock tank that contained laboratory water. The original solution with instars was poured through a metal sieve into another stock tank. The instars collected on the sieve were discarded and the original solution was poured back into the initial stock tank and the corresponding screened insert holding adult daphnids was put back in place. This procedure was then repeated to cull <24 hour old instars for the test.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

76 mg/L (as CaCO3) for 30 mg/L test solutions
78 mg/L (as CaCO3) for 17 mg/L test solutions

Test temperature:

19.5 - 20.5°C

pH:

7.2 - 8.4

Dissolved oxygen:

7.9 - 9.2 mg/L (>86% saturation)

Salinity:

Not determined

Nominal and measured concentrations:

Nominal Concentrations (mg/L) Measured concentrations (mg/L)
Day 0 Day 2 Day 2 (Nominal)
Unfiltered samples Unfiltered samples Filtered samples Unfiltered samples Filtered samples
30.0 28.6 (83.7%) 15.9 8.33 (6.7%) 53 27.8
18.0 28.5 (27.7%) 8.44 6.09 (19%) 46.9 33.8
10.8 25.2 (116%) 4.25 4.20 (24.5%) 39.4 38.9
6.5 5.09 (34.0%) 2.14 2.05 (17.1%) 32.9 31.5
3.9 1.43 (21.6%) 1.27 1.10 (17.1%) 32.6 28.2
2.3 0.85 (25.5%) 0.87 0.80 (2.6%) 37.8 34.8

Details on test conditions:

An intermittent-flow proportional diluter system was designed to deliver six test concentrations, a solvent/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 operated as follows: a precision dosing system (MicroMedic Automatic Pipette System) delivered a specific amount of the test material from a concentrated stock solution to the mixing chamber (5 L) where it was 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 was distributed to the appropriate toxicant cells. When the diluter cycled, the test solution from each primary toxicant cell was mixed with water from its respective water cell and flowed into primary splitting chambers. Three silicone delivery tubes from each primary chamber fed approximately 500 mL to each of three secondary splitting chambers per treatment level. The secondary splitting chambers with ports positioned over 5 holding tubes, contained in each 600 ml borosilicate beaker, delivered approximately 100 mL of test solution into each tube. The replicate beakers had a 2.5 cm2 notch cut in the lip to facilitate water drainage from the vessel. The holding tubes were 2.5 x 12.5 cm with 363 µm mesh bottoms. Each test beaker and tube was labeled with a unique number for identification purposes. The primary , toxicant cells were positioned to deliver in a non-consecutive order of dilution. 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. The temperature of the circulating water bath in the trough was set to maintain 20 ± 1 °C. A temperature controller was used to monitor the temperature during the study period. General operation of the diluter was checked daily using a checklist.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

2.13 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Unfiltered samples

Effect concentrations 2

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

1.93 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Filtered samples

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.27 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Unfiltered samples

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Filtered samples

Details on results:

The 48-hour LC50 values (95% confidence intervals) for the unfiltered and filtered samples were calculated using the day 2 analyses and were determined to be 2.13 (1.13 to 4.56) mg/L and 1.93 (1.65 to 2.26) mg/L respectively. There were no sublethal effects observed. Based on the statistical analysis of the concentration/response data, which took into consideration control mortality, the 48-hour mortality threshold concentrations for unfiltered and filtered samples were 2.14 and 2.05 mg/L, respectively, and the no-observed-effect concentrations (NOEC's) were 1.27 and 1.10 mg/L for the unfiltered and filtered samples.

Results with reference substance (positive control):

There were no mortalities in the DMF control group after 48 hours.

Reported statistics and error estimates:

A computer program was used to calculate the LC50 values and corresponding 95% confidence intervals which were determined with analyzed concentrations. This program has three statistical methods: probit analysis, moving average angle analysis, and binomial probability in 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 is obtained using non-linear interpolation, i.e. log transformation of the concentration and angle transformation of the number dead. Analysis of the concentration/response data was performed using Statistical Analysis System (SAS)® computer software. Specifically, Dunnett's t-test and analysis of variance (ANOVA) were used to determine the no-observed-effect-concentration.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour LC50 values (95% confidence intervals) for the unfiltered and filtered samples were calculated using the day 2 analyses and were determined to be 2.13 (1.13 to 4.56) mg/L and 1.93 (1.65 to 2.26) mg/L respectively. There were no sublethal effects observed. Based on the statistical analysis of the concentration/response data, which took into consideration control mortality, the 48-hour mortality threshold concentrations for unfiltered and filtered samples were 2.14 and 2.05 mg/L, respectively, and the no-observed-effect concentrations (NOEC's) were 1.27 and 1.10 mg/L for the unfiltered and filtered samples.

Executive summary:

A GLP compliant study conducted in accordance with OECD Guideline 202 has been conducted to assess the acute toxicity of the test substance to Daphnia.

 

The 48-hour LC50 values (95% confidence intervals) for the unfiltered and filtered samples were calculated using the day 2 analyses and were determined to be 2.13 (1.13 to 4.56) mg/L and 1.93 (1.65 to 2.26) mg/L respectively. There were no sublethal effects observed. Based on the statistical analysis of the concentration/response data, which took into consideration control mortality, the 48-hour mortality threshold concentrations for unfiltered and filtered samples were 2.14 and 2.05 mg/L, respectively, and the no-observed-effect concentrations (NOEC's) were 1.27 and 1.10 mg/L for the unfiltered and filtered samples.