2-Pyridinecarboxylic acid, 4-(acetylamino)-3,6-dichloro-, methyl ester

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:

31 July 2013 to 2 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

not specified

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentrations in the definitive test were analysed.
- Sampling method: Samples were collected from each bulk test solution at test initiation and the 24-hour renewal and from pooled, spent replicate solutions at 24 hours and test termination. To assess analytical method precision and solution homogeneity, three additional samples were collected on day 0 from the lowest and highest concentration bulk treatment solutions.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Bulk test solutions were made as preparations of a primary stock (100 mg/L, highest dose level). Static renewals were performed after 24 hours and both bulk solution preparations were formed as follows.
A primary stock was prepared by weighing 0.2000 g of test material into a capped scintillation vial. The vial was filled approximately halfway with laboratory dilution water (LDW), capped, shaken vigorously, and poured into a 2 L volumetric flask. This was repeated until no visible test material remained in the vial. The flask was brought to volume with LDW. A stopper was placed in the volumetric flask, and the flask was shaken vigorously at least once while filling and again after bringing it up to volume.
The solution was stirred for approximately 30 minutes at 400 rpm. After stirring, the test material was suspended throughout the solution but was visibly not dissolved. The flask was placed in a water bath sonicator for 80 and 148 minutes for the 0 and 24 h solution preparations, respectively. For both preparations, the stock was sonicated until the solution appeared clear and colourless. The appropriate volume of the primary stock was added to 1 L volumetric flasks and brought to volume with LDW to achieve target concentrations of 3.1, 6.3, 13, 25, and 50 mg/L. The primary stock (100 mg/L) was used as the highest test level and LDW containing no test material was used as the control.
Test solutions were poured into the appropriate test vessel for use in the exposure.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
The test organism was the freshwater daphnid, Daphnia magna, from in-house cultures. Daphnid neonates less than 24 hours old from a laboratory-reared culture were used as the test organisms.
Neonates were only used from daphnids which have had at least 3 broods. The day before neonates were needed for testing (<24 hours prior to test initiation), neonates within the culture vessels were removed and discarded leaving only adult daphnids in the vessels. On the following day, neonates produced overnight in these same culture vessels were again removed from the vessels and placed into a glass beaker containing LDW.
These neonates (<24 hours old) were held in an incubator until used for the study.

TYPICAL CULTURE CONDITIONS
- Temperature: 20 ± 2 °C
- Light (lux): 2050 ± 350
- Photoperiod: 16 hour of light/8 hours of dark
- Water: Laboratory dilution water
- Culture Vessel: 2.9 L glass jars
- Culture Vessel Cover: Plastic wrap
- Culture Vessel Change-Out: At least 3 times weekly
- Feed: Pseudokirchneriella subcapitata and YTC (yeast, wheat grass, Zeigler Finfish starter #1) typically five times weekly. Daphnids were not fed during the test.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

32 to 36 mg CaCO₃/L

Test temperature:

20 to 21 °C

pH:

7.2 to 7.6

Dissolved oxygen:

7.3 to 8.6 mg/L (84 to 99 % saturation)

Nominal and measured concentrations:

Nominal test concentrations: 0 (water control), 3.1, 6.3, 13, 25, 50, and 100 mg/L
Measured test concentrations:

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL glass beakers each containing approximately 40 mL of control or test solution
- Type: Closed. The test vessels were covered to reduce evaporation.
- Renewal rate of test solution (frequency/flow rate): The test solutions were renewed after 24 hours.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
The laboratory dilution water (LDW) was Lake Huron water supplied by the City of Midland Water Treatment Plant. The water was obtained from the upper Saginaw Bay of Lake Huron off Whitestone Point and was limed and flocculated with ferric chloride. The water was pumped to the laboratory prior to municipal treatment for human consumption.
Before use in the laboratory, the water was sand-filtered, pH-adjusted with gaseous CO₂, carbon-filtered, and UV-irradiated.
The water is typically monitored weekly for pH, alkalinity, hardness and conductivity. Periodically, the water is monitored for total organic carbon (TOC), total suspended solids (TSS), and selected inorganic and organic compounds.
- Alkalinity: 22 to 24 mg CaCO₃/L
- Conductivity: 168 to 175 µS/cm

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours of light / 8 hours of dark
- Light intensity: 556 to 689 lux

EFFECT PARAMETERS MEASURED
Daphnids were observed for immobility (inability to swim within approximately 15 seconds after gentle agitation of the test vessel) at approximately 24 and 48 hours of exposure. Any sub-lethal effects (abnormal behaviour or appearance) were recorded.

TEST CONCENTRATIONS
- Range finding study: Yes.
- Test concentrations: Control, 0.1, 1, 10, and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes. Using solutions prepared for the range-finding study, preliminary analytical analyses were performed to determine the definitive test design. The results indicated test solutions should be renewed after approximately 24 hours.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 86.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

86.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

All biological results are expressed in terms of mean measured concentrations of the test material. Summary results of the biological observations are presented in Table 1.
Following 24 hours of exposure, no immobility was observed in any of the test levels. At test termination (48 hours of exposure), immobility was observed in 5, 0, 0, 0, 10, and 15 %, of the daphnids at the 2.36, 4.68, 10.1, 19.9, 40.2, and 86.3 mg/L test levels, respectively.
No immobility was observed at the water control during the conduct of the study. Apart from immobility, no other changes in daphnid behaviour or appearance were observed at any test level.

Reported statistics and error estimates:

Based on the biological observations, neither the Probit nor the Trimmed Spearman-Karber were appropriate methods for analysis as 50 % immobility was not observed in any dose level. The NOEC was determined based on biological interpretation of the data and the highest concentration tested exhibiting no significant daphnid immobility or sublethal effects as determined by a Fisher’s Exact Test.

Any other information on results incl. tables

Table 1: Summary of Results

Mean Measured Test Concentrations (mg/L)	% Immobility
	24 Hours	48 Hours
<LLQ	0	0
2.36	0	5
4.68	0	0
10.1	0	0
19.9	0	0
40.2	0	10
86.3	0	15

LLQ = lowest level quantitated = 0.0390 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity values for Daphnia magna over a 48 h exposure period, based on mean measured concentrations, were EC50 >86.3 mg/L and 48 hour NOEC = 86.3 mg/L.

Executive summary:

A study was conducted to assess the toxicity potential of the test material to the freshwater cladoceran, Daphnia magna in accordance with the standardised guidelines OECD 202 and US EPA OPPTS 850.1010 under GLP conditions.

The study was performed under semi-static conditions for 48 hours with target concentrations of 0 (water control), 3.1, 6.3, 13, 25, 50 and 100 mg /L. Test solutions were renewed after 24 hours.

Five daphnids per replicate were exposed to the test solutions, with 4 replicates per concentration. Test solutions were analysed at test initiation and termination by HPLC/UV. None of the analyses of the water control exhibited a concentration exceeding the lowest level quantitated (LLQ) equivalent to 0.0390 mg /L of the test material.

Mean measured concentrations were: <LLQ, 2.36, 4.68, 10.1, 19.9, 40.2, and 86.3 mg /L.

At test termination (48 hours of exposure), immobility was observed in 5, 0, 0, 0, 10, and 15 %, of the daphnids at the 2.36, 4.68, 10.1, 19.9, 40.2, and 86.3 mg/L test levels, respectively.

No immobility was observed at the water control during the conduct of the study. Apart from immobility, no other changes in daphnid behaviour or appearance were observed at any test level.

The acute toxicity values for Daphnia magna over a 48 h exposure period, based on mean measured concentrations, were EC50 >86.3 mg/L and 48 hour NOEC = 86.3 mg/L.