2,3,5,6-tetrachloropyridine

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24th April 1991 to 24 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 211 (Daphnia magna Reproduction Test)

Deviations:

no

Principles of method if other than guideline:

Study report quotes OECD Guideline 202 in error.

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:

Purity = 98.6%
Identification: AGR 289294
Presumed that lot number WP-900914-748 tested but not stated in report.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Exposure concentrations were analyzed during the 21 day study. Sym-tet standards were prepared. to the range of nominal concentrations in order to quantify the amount of test material in the test solutions. Samples were analyzed on days 0, 2, 5, 12, 19, and 21. Analysis was done on composites of each replicate for each exposure concentration on days 0, 5, 12 and 21. Analysis on each replicate of each exposure concentration was done on days 2 and 19. Initially, the day 0 samples were filtered before analysis because of the evidence of undissolved test material in the test beakers during the acute flow-through toxicity test. However, by day 2 there was no indication of undissolved test material in the test beakers. The chronic test concentrations were an order of magnitude lower than the solubility limit of the test material (in comparison to the acute toxicity test). The remaining analyses were therefore done with unfiltered samples. Once all the daphnids died in a particular replicate, no further analysis of that replicate was performed.

The filtered samples were prepared by taking 8 aliquots from each test beaker and filtering through a 10 mL disposable polypropylene syringe fitted with an Alltech Anotop 25-plus syringe filter (U.20 mm) into 5-dram vials. The standards and 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 nursed auto-sampler vials and analyzed using capillary gas chromatography with electron capture detection (CpGC/ECD).

The amount of test substance 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 of dissolving the test material in aqueous solution, a solvent, dimethyl formamide (DMF), was used as a carrier. Stock solutions of approximately 30,000 m/L were 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 3.0, 1.8, 0.65, 0.39, 0.23 mg/L„ a DMF control (N,N-dimethyl formamide, Fischer Scientific, Lot #902364), and a laboratory water control.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Instars (i.e.daphnids less than 24h old) from a laboratory-reared culture of Daphnia magna were used to initiate the test. Rearing conditions were: illumination (cool white fluorescent) 2500 +/- 110 lux; 16h light / 8h 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 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 insert holding adult daphnids was put back in place. The 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:

21 d

Test conditions

Hardness:

62 -82 mg/L as CaCO3 (for 3 mg/L test solutions) measured weekly during the course of the study.

Test temperature:

20 +/- 1°C

pH:

7.4 to 7.7 (for 3 mg/L test solutions) measured weekly during the course of the study.

Dissolved oxygen:

6.6 to 8.4 mg/l (for 2.67 mg/l measured test solutions) measured weekly during the course of the study.

Salinity:

Not recorded.

Nominal and measured concentrations:

Nominal (mg/L) Mean measured (mg/L)
3 2.67
1.8 1.77
1.08 1.18
0.65 0.61

Mean measured values are an average of measurements taken on days 2, 5, 12, 19 and 21.

Details on test conditions:

Test Procedures:
The test was initiated on June 12, 1991 and terminated on July 3, 1991. Because of the difficulty of dissolving the test material in aqueous solution, a solvent, dimethyl formamide (DMF), was used as a carrier. Stock solutions of approximately 30,000 mg/L were 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 3.0, 1.8, 1.08, 0.65, 0.39 and 0.23 mg/L, a DMF control (N,N-dimethyl formamide, Fischer Scientific, Lot #902364), and a laboratory water control. Twenty instars were exposed to each exposure concentration; one daphnid/tube, five tubes/replicate, four replicates/ concentration. Exposure concentrations were analyzed on days 0, 2, 5, 12, 19, and 21. Daphnids were fed a diet of SeIeneastrum capricornutum at the rate of 2.5 mg dry wt/L of dilution water twice daily. Observations in relation to the fecundity of the organism were recorded each Monday, Wednesday and Friday. Adult mortality and sublethal effects were also recorded. The young produced by each adult were discarded and dead adults removed. An intermittent-flow proportional diluter was used for dosing of the test vessels.

Test Substance Delivery System.
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 dote 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 had a recirculating pump that thoroughly mixed 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. Four silicone delivery tubes from each primary chamber fed approximately 500 mL to each of four secondary splitting chambers per treatment level. The secondary splitting chambers with ports positioned over five 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 366 mm mesh bottoms. Each test beaker and tube was labeled with a unique number for identification purposes. The primary toxicant cols were positioned to deliver in a non-consecutive order of dilution. The diluter wascalibrated 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 24 h. 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.

Physical Analysis.
Measurement of dissolved oxygen (D.O.), pH and temperature was taken from each replicate at least once weekly. The D.O. and pH measurements
were made using Wheaton and Orion Research Meters, respectively. The temperature was taken using a certified ASTM thermometer. The temperature of the circulating water bath in the trough was taken using a . temperature probe in a representative test vessel and was monitored with an Omega temperature recorder. At the beginning and end of the study and once weekly, the controls and the highest exposure concentration with surviving daphnids was characterized by the following water quality parameters: pH, temperature ("C), dissolved oxygen (mg/L), conductivity, tu.mhos/cm), salinity (parts/1000), hardness (mg/LCaCO3), alkalinity (mg/i.. CaCO3), and residual chlorine (ug/L).

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.18 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Effect concentrations 2

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.31 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 3

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1.78 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Effect concentrations 4

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.61 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 5

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

2.48 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 95% CL

Effect concentrations 6

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

1.94 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CL

Effect concentrations 7

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CL

Effect concentrations 8

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.18 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 9

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1.78 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

The calculated values were based on analyzed exposure concentrations. The 2 (48h) and 21-day EC5O values were >2.67, and 2.48 mg/L, respectively. Similarly, the 2 (48h) 48h and 21-day LC50 value were >2.67, and 1.94 mg/L, respectively. The 21-day NOEC and LOEC, for mortality and immobility were not statistically determined but were estimated by observation to be 0.31, 0.61 mg/L (mortality) and. 1.18, 1.78 mg/ L (immobility) in comparison to the control groups. Table 8 lists the sublethal and lethal effects and reproductive status of the dephni.ds during the 2.1-day test period. The first appearance of eggs in the brood sae were observed in the controls by day 9. The first broods in the controls, were observed on day 14. The average cumulative number of young per female in the controls after three broods was greater than 20 and there were no ephippia produced by any of the
organisms. Mortality in the control groups was within the acceptable limits of 20%. The effects on reproduction and growth evaluated by total young per female, brood size per female, total number of broods per female and growth (as length) were based on analyzed exposure concentrations. A
complete set of reproductive and length data is found in Appendix C, The 21-day EC50 values and 95% C.I. as well as the NOEC's and LOEC's for
reproductive and growth end points were calculated by statistical analyses in comparison to the control groups and were as follows: mean total
young per adult, EC50 1.10 (0.21-5.74) mg/L; NOEC 1.18 mg/L; LOEC 1.78 mg/L; mean brood size per adult .1.19 (0.24-5.84) mg/L; NOEC 1.18 mg/L; LOEC 1.78 mg/L; total broods per adult, EC50 1.48 (0.0-3.36) mg/L; NOEC 1.78 mg/L; LOEC 2.67 mg/14 and . growth as length), EC50 >167 mg/L; NOEC 1.78 mg/L, and LOEC 2.6.7 mg/L.

Results with reference substance (positive control):

Not recorded.

Reported statistics and error estimates:

A computer program was used to calculate the EC50 and LC50 values and corresponding 95% confidence intervals for survival. This
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 EC50/LC50 values and its confidence intervals. The binomial method calculates only the confidence interval, while a point estimate of the EC5O/ LC50 is obtained using non¬linear interpolation, i.e., log transformation of the concentration and
angle transformation of the. number dead. The EC50 values for reproduction were estimated by first fitting a line corresponding to the number of young/female on the test concentrations using least squares estimation. Once the equation for the line was determined, the EC50 and its confidence interval were calculated using the method of inverse estimation. The determination of the NOEC and LOEC was made by comparing the treated groups to the control using Dunnett's t-test. This procedute maintained the alpha level at 0.05 while making multiple comparisons to the control group,

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The LC50 values for mortality and the EC50 values for immobility, reproduction, and growth were calculated using mean analyzed exposure
concentrations in a range of 0.25 mg/L to 2.67 mg/L.

Executive summary:

A 21 -day flow-through life-cycle toxicity study on Daphnia magna Straus was conducted according to OECD Guideline 211. The results were as follows:

21-day (mg/L)

Survival LC50 EC50 NOEC LOEC

Immobility 2.48 (2.06 -3.66) 1.18* 1.78*

Mortality 1.94 (1.31 - 4.10) 0.31* 0.61*

Reproduction

Mean Total Young / Adult 1.10 (0.21 - 5.74) 1.18 1.78

Growth

Length in mm >2.67 1.78 2.67

*The NOEC's and LOEC's for survival were not statistically determined but were estimated by observation.