2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 February 2002 to 14 March 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 219 (Sediment-Water Chironomid Toxicity Test Using Spiked Water)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

yes

Details on sampling:

All overlying water, pore water and sediment samples were collected from replicates E through G. During the in-life phase of the definitive study, water samples were removed from one replicate exposure vessel of each treatment level and the control one hour after the test material was added on day 0 (replicate E), on test day 7 (replicate F), and test day 28 (replicate G). Pore water and sediment samples were collected from the 63, 250 and 1000 mg a.i./L treatment levels one hour after application on day 0 (replicate E), and on days 7 (replicate F) and 28 (replicate G). Samples were not removed or analysed from replicate H during this exposure. Overlying water from each replicate vessel was collected by pouring the water into a graduated cylinder. Once the sample was collected, excess water was decanted. Pore water samples were collected by removing the entire sediment sample and centrifuging for 30 minutes at 3000 rpm (approximately 1200 g). The resulting pore water was pipetted from the centrifuge tube. Sediment samples were collected with a stainless steel spatula from the centrifuge tubes, following centrifugation and removal of the pore water sample.
In addition, three quality control (QC) samples were prepared at each sampling interval for each sample matrix (water and sediment), and stored and analysed with the set of study samples. These QC samples were prepared in overlying water and sediment at concentrations of test material similar to the treatment level range. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of exposure solution samples.

Vehicle:

no

Details on sediment and application:

- Stock Solution Preparation
Three individual 1000 mg a.i./L primary stock solutions were prepared by placing 2.1165, 2.1159 and 2.1167 g (2.0000 g as active ingredient) of test material into separate volumetric flasks (2000 mL) and bringing each to final volume with laboratory well water. Each stock was ultrasonicated for two hours and allowed to stir overnight. All volumetric flasks were covered with foil (to avoid light) and stirred at room temperature. Following stirring overnight, the stock solutions were observed to be pale yellow with no undissolved material. Prior to dosing, the three stocks were composited into one stock solution in a 9.5-L glass container and used to dose all treatment levels.

- Test Material Application
Twenty-four hours after the organisms were added to the test vessels, the test material was introduced by adding the appropriate volume of the 1000 mg a.i./L stock solution to the appropriate amount of overlying water. Prior to addition of the stock solution into each vessel, the required amount of overlying water was removed from each test vessel. Following the addition of the stock solution, each exposure solution was gently stirred to aid in the mixing of the test material in the water column prior to resuming aeration. One set of control vessels was prepared for this exposure. Eight dilution water control replicates were prepared using only dilution water with no test material. The control vessels were maintained under the same conditions as the treatment level vessels.

Test organisms (species):

Chironomus riparius

Details on test organisms:

TEST ORGANISM
- Common name: Midge
- Source: Obtained from laboratory cultures maintained at the test site

CULTURE CONDITIONS
Five days prior to test initiation, egg masses were removed from culture aquaria and each individual egg mass was placed in a 30-mL plastic cup with 25 mL of culture water. The egg masses were observed daily until hatching was complete (approximately 24 to 48 hours after release of egg masses by the female midge). Hatched midge larvae were transferred to a shallow glass bowl containing 1 L of culture water and 10 mL of Ankistrodesmus falcatus (4 x 10⁷ cells/mL) to serve as a substrate. Midge larvae were reared under static conditions (20 ± 2 °C) in laboratory well water. During the rearing of the midge larvae, the temperature was 19 °C and the dissolved oxygen ranged from 10.4 to 10.6 mg/L. The culture area was illuminated with fluorescent bulbs at an intensity range of 80 to 90 footcandles (860 to 968 lux). The photoperiod in the culture area was 16 hours of light, 8 hours of darkness. The larvae were reared in the culture bowls for two days after hatching to provide first-instar larvae for use during the exposure to test material. No mortality of midge larvae was observed 48 hours prior to test initiation.
During rearing, the midge larvae were fed a finely-ground suspension of flaked fish food (i.e., 10 mg/mL). Midge larvae were fed daily during the culturing and rearing period.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Test temperature:

19 - 21 °C

pH:

2.8* - 8.1

* The pH of the 500 and 1000 mg a.i./L treatment levels on test days 0 and 1 ranged from 2.8 to 5.6.

Dissolved oxygen:

7.0 - 9.5 mg/L

Nominal and measured concentrations:

63, 130, 250, 500 and 1000 mg a.i./L (nominal)

Details on test conditions:

OVERLYING WATER
Overlying water used during this study was laboratory well water from the same source as the culture water. The water used during the definitive exposure was characterised as having total hardness and total alkalinity ranged as calcium carbonate (CaCO₃) of 46 to 52 and 34 to 35 mg/L, respectively, a pH range of 7.0 to 7.3, and a specific conductivity range of 150 to 160 micromhos per centimetre (μmhos/cm).

SEDIMENT
Artificial sediment was the substrate used in the exposure. The artificial sediment was prepared according to OECD Guideline No. 207 by mixing the following components (based on dry weight): 1.66 kg sphagnum peat, 4.0 kg kaolin clay and 14.34 kg industrial sand (i.e., 8.3, 20 and 71.7 %, respectively). The dry components were blended together in a large-scale laboratory mixer and a small amount of overlying water was added to moisten the sediment before it was added to the exposure vessels. The artificial sediment used in the study was characterised as having an organic carbon content of 1.8 %, a particle size distribution of 77 % sand, 6 % silt and 17 % clay, a pH of 7.5, and a moisture content at 1/3 bar (water holding capacity prior to use in the study) of 11.3 %. Prior to use, the artificial sediment was placed in an aquarium and clean laboratory well water was provided on a continuous basis for one week to condition the sediment.

TEST SYSTEM
- Test container: 600-mL clear glass beakers
- Sediment volume: 75 mL
- Weight of wet sediment: 134 g (99 g on a dry weight basis)
- Overlying water volume: 300 mL
- Depth of sediment: 1.5 cm
- Aeration: yes
- Aeration frequency and intensity: At the time of addition of the midge larvae and for the 24 hours following, aeration of the water was suspended. 24 hours after the test organisms were added, and following application of the test material to the test vessels, aeration was resumed at 1 to 3 bubbles per second.
- Replacement of evaporated test water, if any: no. Each test vessel was covered with a clear plastic plate to minimise evaporation and trap emerging adult midge.

EXPOSURE REGIME
- Feeding regime: Prior to introducing the midge to the test vessels, 0.50 mL of finely ground flaked fish food suspension (10 mg/mL) was added to each test vessel. Test midges were fed 0.50 mL of finely ground flaked fish food suspension (10 mg/mL) daily, on test days -1 through 10. On days 11 through termination, midges were fed 1.0 mL of finely ground flaked fish food suspension (10 mg/mL) daily.

OTHER TEST CONDITIONS
- Light quality: The test area was illuminated with fluorescent bulbs
- Photoperiod: 16 hours of light / 8 hours of darkness
- Light intensity: 50 to 80 footcandles (538 to 861 lux)

EFFECT PARAMETERS MEASURED
Test Monitoring
Replicate vessels A - D were examined at test initiation and daily thereafter, until test termination (day 28). Observations of midge emergence and abnormal behaviour were made and the physical characteristics of the test solutions were recorded. Starting on test day 10 and during the period of expected emergence, a daily check of emerged midges was made. The sex and number of adult midge that emerged daily were recorded. Male midge were identified by their plumose antennae. The development rate of male, female, and male and female midge combined, in each exposure vessels was also determined.

Water Quality Measurements
Water quality measurements made during the study were performed in the four replicate exposure vessels (A through D) established for monitoring the biological performance of the exposed midge. Measurements of dissolved oxygen concentration, temperature and pH were made on the day the test organisms were added (day -1), the day of test material application (day 0), test day 1, and at test termination (day 28) in each exposure vessel. In addition, dissolved oxygen concentration and temperature were measured daily in each replicate vessel of each treatment level and the control during the 28-day exposure. The temperature was continuously monitored in replicate H of the 250 mg a.i./L treatment vessel throughout the study. Total hardness, alkalinity, specific conductivity, and total ammonia of the test solutions were determined at test initiation and at test termination in a composite sample (replicates E through H at initiation, and A through D at termination) from the highest treatment level and control solution.

VEHICLE CONTROL PERFORMED: yes

PRELIMINARY TESTING
Prior to initiating the definitive study, a preliminary range-finding exposure was conducted at nominal test material levels of 0.10, 1.0, 10, 100 and 1000 mg a.i./L and a dilution water control. Three replicates of twenty midge (2 days old) were exposed to each treatment level and control. Following 24 days of exposure, the mean percent emergence among midge exposed to the nominal concentrations tested (0.10, 1.0, 10, 100 and 1000 mg a.i./L) was 82, 70, 83, 85 and 0 %, respectively. During the same period, mean percent emergence among the midge exposed to the control was 82 %. The mean development rate for the 1000 mg a.i./L treatment level could not be determined as no emergence was observed in this treatment level. The mean development rate (male/female midge combined), after 24 days of exposure, for the remaining nominal concentrations tested (0.10, 1.0, 10, 100 mg a.i./L) was 0.0588, 0.0636, 0.0632 and 0.0596, respectively. During the same period, mean development rate among the midge exposed to the control was 0.0604. Based on these results, the following nominal test material concentrations were selected for the definitive study: 63, 130, 250, 500 and 1000 mg a.i./L.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

680 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Remarks on result:

other: 640 - 720 mg a.i./L (95% CL)

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

130 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Details on results:

Following 28 days of exposure, midge percent emergence and mean development rate for male, female, and male/female combined in the control was 94 % and 0.0625, 0.0546 and 0.0581, respectively.
At test termination (day 28), the mean percent emergence observed among the organisms exposed to the three highest nominal treatment levels (250, 500 and 1000 mg a.i./L) was 80, 75 and 0 % and was statistically different from the mean percent emergence of the control organisms (94 %). The mean percent emergence observed among the organisms exposed to the remaining nominal treatment levels (63 and 130 mg a.i./L) was 88 and 86 %, respectively and was not statistically different from the mean percent emergence of the control organisms (94 %).
The mean development rates were not determined for the 1000 mg a.i./L treatment level as no midge (male or female) emerged at this treatment level. The mean development rate among male midge emerged in the remaining nominal treatment levels (63, 130, 250 and 500 mg a.i./L) was 0.0590, 0.0611, 0.0598 and 0.0582 respectively. Mean development rate of male midge in the 500 mg a.i./L was statistically different from the mean development rate of the male control organisms (0.0625).
The mean development rate among female midge emerged in the nominal treatment levels (63, 130, 250 and 500 mg a.i./L) was 0.0557, 0.0537, 0.0555 and 0.0522, respectively, and was not statistically different from the mean development rate of the female control organisms (0.0546).
The mean development rate for male and female midge (combined) emerged in the nominal treatment levels (63, 130, 250 and 500 mg a.i./L) was 0.0572, 0.0570, 0.0579 and 0.0557, respectively, and was not statistically different from the mean development rate of the male and female (combined) control organisms (0.0581).

Reported statistics and error estimates:

At the termination of the study, data obtained on midge emergence and development rate were statistically analysed to identify significant treatment-related effects. The LOEC and the NOEC were determined. Analyses were performed using the mean replicate organism response in each treatment group. All statistical analyses were conducted at the 95 % level of certainty except in the case of the Shapiro-Wilks Test and the Bartlett's Test, in which the 99 % level of certainty was applied. The following procedures were used:
1. Determination of adverse effects on the percent emergence was determined after transformation (e.g., arcsine square-root percentage) of the data.
2. Shapiro-Wilks Test for normality (Weber et al., 1989) was conducted to compare the observed sample distribution with a normal distribution for all endpoints. The assumption that observations are normally distributed must be validated before subsequent analyses, following parametric procedures, can be performed. If the data are not normally distributed, then a non-parametric procedure is used for subsequent analyses. Analysis of percent emergence and development rate data met this assumption of normal distribution.
3. As a check on the assumption of homogeneity of variance implicit in parametric statistics, data for each endpoint were analysed using Bartlett's Test (Sokal and Rohlf, 1981). Percent emergence and development rate data passed the test for homogeneity.
4. For this study, the percent emergence and development rate data met the assumptions for normal distribution and homogeneity, therefore, Williams' Test was used to establish treatment effects on these endpoints.
A computer program (Gulley, et al., 1989) was used to perform the computations.

EC50 Calculation
The nominal concentrations tested were used to estimate the EC50 and 95 % confidence intervals. A computer program was used to determine the EC50 values using the linear interpolation method (Norberg-King, 1993).

Analytical Chemistry

Analysis of the stock solution (1000 mg a.i./L) used to apply the test material to the overlying water resulted in a measured concentration of 100 % of the nominal concentration. This result established that the appropriate mass of test material was initially introduced to the overlying water during the application procedure.

In the overlying water samples, measured concentrations of test material at 1-hour in the 63, 130, 250, 500 and 1000 mg a.i./L treatment levels were 61, 130, 240, 530 and 920 mg a.i./L, respectively. On test day 7, measured concentrations of test material in the 63, 130, 250, 500 and 1000 mg a.i./L treatment levels, were 58, 120, 260, 560 and 1060 mg a.i./L, respectively. On test day 28, (test termination), measured concentrations of test material in the 63, 130, 250, 500 and 1000 mg a.i./L treatment levels were 55, 120, 240, 470 and 940 mg a.i./L, respectively.

In the pore water samples, measured concentrations of test material at 1-hour in the 63, 250 and 1000 mg a.i./L treatment levels were 11, 44 and 170 mg a.i./L, respectively. On test day 7, measured concentrations of test material in the 63, 250 and 1000 mg a.i./L treatment levels increased to 51, 210 and 850 mg a.i./L, respectively. On test day 28 (test termination), measured concentrations of test material in the 63, 250 and 1000 mg a.i./L treatment levels were 53, 230 and 930 mg a.i./L, respectively.

In the sediment samples, measured concentrations of test material at 1-hour in the 63, 250 and 1000 mg a.i./L treatment levels were 5.6, 17 and 150 mg a.i./kg, respectively. On test day 7, measured concentrations of test material in the 63, 250 and 1000 mg a.i./L treatment levels increased to 24, 88 and 400 mg a.i./kg, respectively. On test day 28 (test termination), measured concentrations of test material in the 63, 250 and 1000 mg a.i./L treatment levels were 10, 91 and 680 mg a.i./kg, respectively.

Based on the analytical results of this study, test material concentrations in the overlying water remained consistent throughout the exposure period (i.e., 90 to 100 % of the nominal concentrations), and established that it did not partition to the sediment in significant quantities.

Water Quality Parameters

Initial pH measurements on days 0 and 1 in the two highest treatment levels tested, 500 and 1000 mg a.i./L ranged from 2.8 to 5.6 and were significantly lower than the control pH measurements at the same intervals (i.e., 7.5 to 8.1). The low pH in the two highest treatment levels is due to the concentration of test material in the solutions. The remaining water quality parameters were unaffected by the concentrations of test material tested and remained within acceptable limits. Daily measurements of the temperature in the exposure solutions, and continuous monitoring in replicate H of the 250 mg a.i./L test solution established a temperature range of 18 to 21 °C throughout the definitive study.

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations applied and midge emergence, the Lowest-Observed- Effect Concentration (LOEC) was established to be 250 mg a.i./L and the No-Observed-Effect Concentration (NOEC) was established to be 130 mg a.i./L. The 28-day EC50, based on midge emergence, was calculated to be 680 mg a.i./L (with 95 % corresponding confidence intervals of 640 to 720 mg a.i./L).

Executive summary:

The toxicity of the test material to the sediment dwelling midge, Chronomus riparius, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 219.

During the study the midge were exposed to nominal concentrations of test material of 63, 130, 250, 500 and 1000 mg a.i./L, and a dilution water control, under static conditions, for a period of 28 days. Eight replicate vessels were prepared for each test material treatment level and control; 20 midge larvae (2 days old) were added to each replicate test vessel (A through D), the additional replicates (E through H) were established for chemical analysis of the overlying water, pore water and sediment.

During the exposure period, replicate vessels A through D were observed daily for midge emergence and abnormal behaviour; physical characteristics of the test solutions were also recorded. Starting on test day 10 and during the period of expected emergence (typically starting at day 13 to 16 and lasting until day 25), a daily check of emerged midges was made. The sex and number of adult midge that emerged daily were recorded. Male midge were identified by their plumose antennae. The development rate of male, female, and male and female midge combined, in each exposure vessels was also determined.

A low pH was recorded in the two highest treatment levels (2.8 - 5.6); however, the remaining water quality parameters were unaffected by the concentrations of test material tested and remained within acceptable limits.

Chemical analyses of the water and sediment revealed test material concentrations in the overlying water remained consistent throughout the exposure period (i.e., 90 to 100 % of the nominal concentrations), and established that it did not partition to the sediment in significant quantities.

Following 28 days of exposure, midge percent emergence and mean development rate for male, female, and male/female combined in the control was 94 % and 0.0625, 0.0546 and 0.0581, respectively. At test termination (day 28), the mean percent emergence observed among the organisms exposed to the three highest nominal treatment levels (250, 500 and 1000 mg a.i./L) was 80, 75 and 0% and was statistically different from the mean percent emergence of the control organisms (94 %). The mean percent emergence observed among the organisms exposed to the remaining nominal treatment levels (63 and 130 mg a.i./L) was 88 and 86 %, respectively and was not statistically different from the mean percent emergence of the control organisms (94 %).

The mean development rates were not determined for the 1000 mg a.i./L treatment level as no midge (male or female) emerged at this treatment level. The mean development rate among male midge emerged in the remaining nominal treatment levels (63, 130, 250 and 500 mg a.i./L) was 0.0590, 0.0611, 0.0598 and 0.0582 respectively. Mean development rate of male midge in the 500 mg a.i./L was statistically different from the mean development rate of the male control organisms (0.0625).

The mean development rate among female midge emerged in the nominal treatment levels (63, 130, 250 and 500 mg a.i./L) was 0.0557, 0.0537, 0.0555 and 0.0522, respectively, and was not statistically different from the mean development rate of the female control organisms (0.0546). The mean development rate for male and female midge (combined) emerged in the nominal treatment levels (63, 130, 250 and 500 mg a.i./L) was 0.0572, 0.0570, 0.0579 and 0.0557, respectively, and was not statistically different from the mean development rate of the male and female (combined) control organisms (0.0581).

Based on the nominal concentrations applied and midge emergence, the Lowest-Observed- Effect Concentration (LOEC) was established to be 250 mg a.i./L and the No-Observed-Effect Concentration (NOEC) was established to be 130 mg a.i./L. The 28-day EC50, based on midge emergence, was calculated to be 680 mg a.i./L (with 95 % corresponding confidence intervals of 640 to 720 mg a.i./L).

Description of key information

28 day EC50 (midge emergence) = 680 mg a.i./L; NOEC = 130 mg a.i./L,  Chronomus riparius, OECD 219, Putt (2002)

Additional information

In the key study reported by Putt (2002) the toxicity of the test material to the sediment dwelling midge, Chronomus riparius, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 219. The study was assigned a reliability score of 1 in line with the principles

During the study the midge were exposed to nominal concentrations of test material of 63, 130, 250, 500 and 1000 mg a.i./L, and a dilution water control, under static conditions, for a period of 28 days. Eight replicate vessels were prepared for each test material treatment level and control; 20 midge larvae (2 days old) were added to each replicate test vessel (A through D), the additional replicates (E through H) were established for chemical analysis of the overlying water, pore water and sediment.

During the exposure period, replicate vessels A through D were observed daily for midge emergence and abnormal behaviour; physical characteristics of the test solutions were also recorded. Starting on test day 10 and during the period of expected emergence (typically starting at day 13 to 16 and lasting until day 25), a daily check of emerged midges was made. The sex and number of adult midge that emerged daily were recorded. Male midge were identified by their plumose antennae. The development rate of male, female, and male and female midge combined, in each exposure vessels was also determined.

A low pH was recorded in the two highest treatment levels (2.8 - 5.6); however, the remaining water quality parameters were unaffected by the concentrations of test material tested and remained within acceptable limits.

Chemical analyses of the water and sediment revealed test material concentrations in the overlying water remained consistent throughout the exposure period (i.e., 90 to 100 % of the nominal concentrations), and established that it did not partition to the sediment in significant quantities.

Following 28 days of exposure, midge percent emergence and mean development rate for male, female, and male/female combined in the control was 94 % and 0.0625, 0.0546 and 0.0581, respectively. At test termination (day 28), the mean percent emergence observed among the organisms exposed to the three highest nominal treatment levels (250, 500 and 1000 mg a.i./L) was 80, 75 and 0% and was statistically different from the mean percent emergence of the control organisms (94 %). The mean percent emergence observed among the organisms exposed to the remaining nominal treatment levels (63 and 130 mg a.i./L) was 88 and 86 %, respectively and was not statistically different from the mean percent emergence of the control organisms (94 %).

The mean development rates were not determined for the 1000 mg a.i./L treatment level as no midge (male or female) emerged at this treatment level. The mean development rate among male midge emerged in the remaining nominal treatment levels (63, 130, 250 and 500 mg a.i./L) was 0.0590, 0.0611, 0.0598 and 0.0582 respectively. Mean development rate of male midge in the 500 mg a.i./L was statistically different from the mean development rate of the male control organisms (0.0625).

The mean development rate among female midge emerged in the nominal treatment levels (63, 130, 250 and 500 mg a.i./L) was 0.0557, 0.0537, 0.0555 and 0.0522, respectively, and was not statistically different from the mean development rate of the female control organisms (0.0546). The mean development rate for male and female midge (combined) emerged in the nominal treatment levels (63, 130, 250 and 500 mg a.i./L) was 0.0572, 0.0570, 0.0579 and 0.0557, respectively, and was not statistically different from the mean development rate of the male and female (combined) control organisms (0.0581).

Based on the nominal concentrations applied and midge emergence, the Lowest-Observed- Effect Concentration (LOEC) was established to be 250 mg a.i./L and the No-Observed-Effect Concentration (NOEC) was established to be 130 mg a.i./L. The 28-day EC50, based on midge emergence, was calculated to be 680 mg a.i./L (with 95 % corresponding confidence intervals of 640 to 720 mg a.i./L).