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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 February 2002 to 12 February 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 72-3 (Estuarine/Marine Fish, Mollusk, or Shrimp Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1025 (Bivalve Acute Toxicity (shell deposition test))

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

yes

Details on sampling:

One replicate of the high, middle and low treatment levels and the dilution water control were sampled twice prior to the start of the definitive exposure and analysed for the concentration of test material present in each vessel. Results of these pre-test analyses were used to judge whether sufficient quantities of test material were being delivered and maintained in the exposure aquaria to initiate the oyster shell deposition exposure. In addition, a sample of the stock solution used to prepare the exposure solutions was analysed twice prior to test initiation.
During the definitive study, samples were removed from one replicate solution of each treatment level and the controls at each sampling interval. At test initiation replicate A was sampled and at test termination replicate B was sampled and analysed for test material concentration. Stock solution analyses were also performed. Three quality control (QC) samples were prepared at each sampling interval and remained with the set of exposure solution samples throughout the analytical process. 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:

yes

Details on test solutions:

PREPARATION OF TEST SOLUTION
Stock solutions (200 mg a.i./mL) were prepared during the study by placing approximately 212 g (200 g as active ingredient) of test material in a 1000-mL volumetric flask and diluting to volume with dimethylformamide (DMF). The resultant stock solution was observed to be clear with a dark amber colour.
The solvent control stock solution (0.40 mL/mL) was prepared by placing 400 mL of DMF in a volumetric flask and diluting to a volume of 1000 mL with distilled water.

APPLICATION OF TEST SOLUTION
A Harvard peristaltic pump was calibrated to deliver 0.1875 mL/minute of the 200 mg a.i./mL stock solution to the diluter system's mixing chamber which also received 0.375 L/min of dilution water. The mixing chamber was positioned within an ultrasonic water bath where the contents were continuously sonicated and stirred, using a water-driven magnetic stir plate, to aid in the solubilisation of the test material. The concentration of the test material in the solution contained within the mixing chamber was equivalent to that of the highest nominal concentration tested (100 mg a.i./L) and was subsequently diluted (60 % dilution factor) to provide the remaining nominal test concentrations: 60, 36, 22 and 13 mg a.i./L.
The same pump delivered 0.1875 mL/minute of the solvent control stock solution (0.40 mL/mL) to 0.150 L of dilution water per minute which was subsequently delivered to the solvent control vessels. The solvent control concentration was 0.50 mL/L, which was equivalent to the concentration of solvent present in the highest test concentration. A set of control vessels was also established which contained the same dilution water and was maintained under the same conditions as the treatment level vessels, but contained no test material or solvent.
The diluter system was calibrated to deliver approximately 75 mL/minute of exposure solution to each replicate test vessel.

Test organisms (species):

other aquatic mollusc: Crassostrea virginica

Details on test organisms:

TEST ORGANISM
- Common name: Eastern oysters
- Health upon arrival: If any oyster appeared unsuitable for testing due to the presence of boring sponges and/or mudworms, it was discarded. In addition, several oysters were opened and carefully examined to confirm that no parasites were present, then discarded. Oysters were determined to be reproductively immature by pressing the tissue in the area where gametes are stored to confirm that none were present.
- Age at study initiation: The oysters were of similar age and had a mean valve height of 39 ± 4 mm (N = 30)
- Feeding during test: During the exposure, the oysters received supplemental feedings of algae (Tetraselmus maculata). Concentrated volumes of algal suspension (approximately 10⁷ cells/mL) were added to each test aquarium three times daily to maintain an average concentration of approximately 10⁵ cells/mL in the test solutions during the four-day exposure.

ACCLIMATION
- Acclimation period: 13 days
Oysters were held in a polypropylene tray through which seawater flowed continuously at the test laboratory for 13 days prior to testing. During this acclimation period, the seawater in which the oysters were held had a temperature range of 17 to 21 °C, a pH range of 7.0 to 7.9 and a dissolved oxygen concentration range of 71 to 110 % of saturation. At their source, the oysters were collected from water which had a salinity of approximately 15 ‰. During the acclimation period at the test laboratory, the salinity was gradually increased from 17 to 34 ‰. During the culture period, oysters were fed a supplementary algal diet of Tetraselmus maculata prepared in seawater from a concentrate. Less than 1 % mortality was observed among the oyster population during the seven days before test initiation.
Prior to testing, 3 to 5 mm of the new peripheral shell growth of each oyster were removed by grinding the shell to a blunt edge using a fine-grit grinding wheel. Approximately ten oysters, in addition to the number required for testing, were prepared; any oysters which appeared less than optimal were discarded.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

20 - 21 °C

pH:

7.2 - 8.0

Dissolved oxygen:

5.7 - 7.8 mg/L

Salinity:

32 - 33 ‰

Nominal and measured concentrations:

13, 22, 36, 60 and 100 mg a.i./L (nominal)
12, 21, 31, 50 and 89 mg a.i./L (mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: Test vessels consisted of glass aquaria measuring 49.5 x 25.5 x 29 cm and were equipped with an overflow side drain positioned at a height of 14 cm which maintained a test solution volume of approximately 18 L. The flow of exposure solution to each aquarium (75 mL/minute) provided approximately 6.0 solution volume replacements every 24 hours. In addition, the contents of each aquarium were continuously circulated. Circulation was provided by pumping the exposure solution with a magnetic drive pump from one end of the aquarium and returning it to the opposite end. Return water flowed through a perforated Teflon® tube, which extended the length of the aquarium. The flow rate of the recirculating test solution was 0.075 L/minute or about 5.25 L per oyster per hour. The circulating system aided in evenly distributing the algae fed to the oysters and in mixing the flow of test solution throughout each aquarium.
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2

DILUTION WATER
Natural unfiltered seawater was used as dilution and control water during this study. Seawater was pumped from the Cape Cod Canal, Bourne, Massachusetts from about 1 to 4 m offshore at a depth of approximately 0.5 m. The seawater was then transferred by a pump (fibreglass reinforced thermoplastic housing) through polyvinyl chloride (PVC) pipes and transported to the laboratory in a 3400-L fibreglass holding tank. In the laboratory the seawater was recirculated within an epoxy-coated concrete holding reservoir prior to use. The seawater was pumped under constant pressure through PVC pipes to the constant flow diluter system.
The seawater used for this study had a salinity of 32 ‰ and a pH of 8.0 (based on 0-hour measurements of the dilution water control solutions). Representative samples of the dilution water source were analysed periodically for the presence of pesticides, PCBs and toxic metals. None of these compounds were detected in any of the water samples analysed, in agreement with ASTM (2000) standard practices.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: A photoperiod of 16 hours of light and 8 hours of darkness was maintained throughout the test. Sudden transitions from light to dark and vice versa were avoided.
- Light intensity: No data; illumination was provided by Sylvania Octron® fluorescent bulbs.

EFFECT PARAMETERS MEASURED
Biological observations (e.g., visible abnormalities, such as excessive mucous production or a failure to siphon and feed, as evidenced by a lack of faecal and pseudofaecal production) and observations of the physical characteristics of the test solutions were made at test initiation and at each subsequent 24-hour interval until termination of the test. Sub-lethal effects were determined by a comparison of the performance and appearance of the exposed oysters to that of the control oysters. After 96 hours of exposure, the oysters were removed from the test aquaria and the new shell growth was measured microscopically to the nearest 0.1 mm using a calibrated micrometer.

ENVIRONMENTAL PARAMETERS
During the definitive exposure, the pH, temperature, salinity and dissolved oxygen concentration were measured daily in each replicate aquarium. The salinity concentrations were measured with a Vee Gee Scientific refractometer; pH was measured with a Hanna HI92ION pH meter and combination electrode; dissolved oxygen concentration was measured with a YSI Model No. 57 dissolved oxygen meter and probe, and the daily temperature was measured with a Fisher alcohol thermometer. In addition, temperature was monitored continuously in one replicate (A) of the 100 mg a.i./L solution using a Fisher Scientific Minimum/Maximum Thermometer.

PRELIMINARY TESTING
Prior to initiating the definitive study, a preliminary flow-through acute test was conducted. Eastern oysters were exposed to nominal test material concentrations of 2.6, 6.4, 16, 40 and 100 mg a.i./L, and a dilution water control. One aquarium containing 20 oysters was established for each test concentration and the control. Following 96 hours of exposure, 16, 14, 13, 13 and 42 % reduction in shell growth was observed among oysters exposed to the 2.6, 6.4, 16, 40 and 100 mg a.i./L treatment levels, respectively. Based on the results of preliminary testing, nominal test concentrations of 13, 22, 36, 60 and 100 mg a.i./L were selected for definitive testing.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 89 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: shell growth

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

89 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: shell growth

Details on results:

Growth among dilution water control oysters at test termination averaged 2.7 mm, and the growth for the solvent control oysters averaged 2.6 mm. The growth of both control groups during this study was within the historical range of the laboratory. Based on these data, the amount of shell deposition observed during this study is considered representative for this species and acceptable for establishing the relative toxicity of the test material to Eastern oysters.
At test termination, no mortality was observed among oysters at any of the treatment levels tested. The percent reduction in shell growth was 12 % for oysters exposed to the highest mean measured concentration tested (89 mg a.i./L). The remaining treatment levels (12, 21, 31 and 50 mg a.i./L) all exhibited a positive response compared to the pooled controls. No sublethal effects were observed among any of the exposed oysters throughout the test concentration range.

Reported statistics and error estimates:

The 96-hour EC50 value and 95 % confidence intervals were determined by fitting the untransformed or transformed (i.e., growth data as percent reduction transformed to probit, concentrations transformed to log concentration) data to a best fit linear regression curve based on least squares. Thus, a total of four linear regression curves were computed. The regression line which provided the best fit of the untransformed or transformed data was selected based on the highest associated coefficient of determination, i.e., r². This regression equation was then applied to calculate the EC50 and its 95 % confidence intervals, using the method of inverse prediction (Sokal and Rohlf, 1981). A computer program was developed at the test laboratory to assist in these computations. Results reported are based on the mean measured concentrations of the test material.
The No-Observed-Effect Concentration (NOEC) for the 96-hour exposure period is statistically determined by using Williams' Test. The NOEC is defined as the highest concentration at, and below which, there is no statistically significant reduction in growth.

Analytical Chemistry

Based on the stock solution consumption rates and the twice daily check of diluter calibration, it was determined that the diluter system, which prepared and delivered the test solutions to the exposure aquaria, functioned properly during the pre-test period and throughout the 96-hour definitive study.

Throughout the exposure period, all mixing chamber, splitter and aquarium solutions were observed to be clear and colourless with no visible evidence of undissolved test material. Analysis of the stock solution (200 mg a.i./mL) used to prepare test concentrations resulted in measured concentrations that were 106 and 105 % of the nominal concentration at 0 hour and 96 hour, respectively. The results of the analysis of the exposure solutions for test material during the in-life portion of the definitive exposure, in terms of mean measured exposure concentrations, were 12, 21, 31, 50 and 89 mg a.i./L, which averaged 95, 97, 87, 84 and 89 % of nominal, respectively.

Water Quality Parameters

The pH decreased with increasing test concentrations. The remaining water quality parameters measured were unaffected by the concentrations of test material tested and remained within acceptable levels for the survival and growth of Eastern oysters. Daily monitoring of the test solutions and continuous temperature monitoring established that the exposure solution temperature was maintained at 20 to 21 °C during the exposure period.

Table 1: Effects of the Test Material on Shell Deposition After 96 Hours of Exposure

Mean measured conc. (mg a.i./L)	Mean shell deposition (mm)	Mean percent reduction
Control	2.7	NA
Solvent control	2.6	NA
Pooled control	2.7	NA
12	2.9	0
21	2.9	0
31	3.1	0
50	2.8	0
89	2.4	12

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 96-hour EC50 was empirically estimated to be greater than 89 mg a.i./L. No statistically significant difference was determined at any treatment level. Therefore, the No-Observed-Effect Concentration (NOEC) was determined to be 89 mg a.i./L.

Executive summary:

The acute toxicity of the test material to Eastern oysters was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 72-3 and EPA OPPTS 850.1025.

During the study, oysters were exposed to nominal test concentrations of test material of 13, 22, 36, 60 and 100 mg a.i./L, under flow-through conditions. Dilution water and solvent (DMF) control treatments were included. All treatment levels and the controls were maintained in duplicate. The exposure of oysters was initiated by impartially selecting and placing 20 oysters in each test aquarium (40 per treatment level and the controls). Oysters were spaced equidistant from one another with their valve inflow openings facing toward the flow of water from the Teflon circulator tube. At 24, 48, 72 and 96 hours the oysters were observed for mortality and visible abnormalities. Sub-lethal effects were determined by a comparison of the performance and appearance of the exposed oysters to that of the control oysters. After 96 hours of exposure, the oysters were removed from the test aquaria and the new shell growth was measured microscopically to the nearest 0.1 mm using a calibrated micrometer. During the definitive exposure, the pH, temperature, salinity, and dissolved oxygen concentration were measured daily in each replicate aquarium.

Growth among dilution water control oysters at test termination averaged 2.7 mm, and the growth for the solvent control oysters averaged 2.6 mm. The growth of both control groups during this study was within the historical range of the laboratory. Based on these data, the amount of shell deposition observed during this study is considered representative for this species and acceptable for establishing the relative toxicity of the test material to Eastern oysters. At test termination, no mortality was observed among oysters at any of the treatment levels tested. The percent reduction in shell growth was 12 % for oysters exposed to the highest mean measured concentration tested (89 mg a.i./L). The remaining treatment levels (12, 21, 31 and 50 mg a.i./L) all exhibited a positive response compared to the pooled controls. No sublethal effects were observed among any of the exposed oysters throughout the test concentration range.

Therefore, under the conditions of the study, the 96-hour EC50 was empirically estimated to be greater than 89 mg a.i./L (based on mean measured concentrations). The No-Observed-Effect Concentration (NOEC) was determined to be 89 mg a.i./L.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 February 2002 to 18 February 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 72-3 (Estuarine/Marine Fish, Mollusk, or Shrimp Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1035 (Mysid Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

yes

Details on sampling:

During the 96-hour definitive study, one sample was removed from each treatment level and the controls at each sampling interval. At test initiation, samples were removed from the intermediate vessel prior to division into replicate test vessels, and at test termination samples were removed from a composite of replicate A and B, and analysed for test material concentrations. Three quality control (QC) samples were prepared at each sampling interval and remained with the set of exposure solution samples through the analytical process. 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:

yes

Details on test solutions:

A 0.10 mg a.i./mL stock was prepared by placing 0.6354 g of test material (0.600 g as active ingredient) in a flask and diluting to 6000 mL with natural seawater adjusted to a salinity of 20 ± 3 ‰. The natural seawater was previously spiked with dimethylformamide (DMF) at a concentration of 0.10 mL/L. The stock solution was observed to have a slight yellow tint. Exposure solutions were prepared by diluting the 0.10 mg a.i./mL stock solution with the previously spiked natural filtered seawater adjusted to a salinity of 20 ± 3 ‰. Preparation of exposure solutions in this manner allowed equal concentrations of solvent (0.10 mL/L) in all treatments.
The control solution was natural filtered seawater adjusted to a salinity of 20 ± 3 ‰ without the addition of test material or solvent (DMF).
A 0.10 mL/L solvent control solution was prepared by placing 0.800 mL of DMF in a flask and diluting to 8000 mL with natural seawater (20 ± 3 ‰). This solution was also used (6000 mL) for dilution water.

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
- Source: The mysids used in the toxicity test were obtained from laboratory cultures maintained at the test laboratory
- Age at study initiation: < 24 hours old
- Method of culturing: Mysids were cultured in one of several 76-L glass aquaria with a closed-loop recirculating filtration system providing artificial filtered seawater to the aquaria. The seawater in the aquaria was characterised as having a salinity range of 23 to 26 ‰ for the 14-day period prior to initiation of the definitive exposure. The area in which the mysids were cultured received a regulated photoperiod of 16 hours of light and 8 hours of darkness. Commercial aquarium heaters were used to maintain the culture solution temperature at 25 ± 2 °C. Mysids were fed live brine shrimp (Artemia salina) nauplii, ≤ 48 hours old (post-hydration), twice daily.
- Feeding during test: Yes. Live brine shrimp nauplii (Artemia salina) were added to each test vessel containing live test organisms once daily during the exposure period.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

24 - 25 °C

pH:

6.8 - 7.9

Dissolved oxygen:

6.2 - 8.1 mg/L

Salinity:

21 ‰

Nominal and measured concentrations:

0, 13, 22, 36, 60 and 100 mg a.i./L (nominal)
0, 14, 22, 36, 59, 100 mg a.i./L (mean measured concentrations)

Details on test conditions:

TEST SYSTEM
- Test vessel: 1 L glass beakers
- Type ( open / closed): No data
- Fill volume: 900 mL of test solution
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water used during this study was prepared by filtering natural seawater collected from the Cape Cod Canal, Bourne, Massachusetts. The seawater was transferred with a pump (fibreglass reinforced thermoplastic housing) and a polyvinyl chloride (PVC) pipe and was then transported to the laboratory in a 3400-L fibreglass tank. In the laboratory, the seawater was passed through a series of polypropylene core filters (20- and 5-micron) and then recirculated within an epoxy-lined concrete reservoir prior to use. The seawater was pumped to the laboratory under constant pressure through PVC pipe and a polypropylene heat exchanger system. The salinity of the natural seawater was adjusted to 20 ± 3 ‰ with laboratory well water prior to use.
- Total organic carbon: < 2.0 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: The dilution water was analysed at the test laboratory periodically to check for presence of pesticides, PCBs and toxic metals. None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analysed.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours of light / 8 hours of darkness
- Light intensity: 60 to 80 footcandles at the solutions’ surface

EFFECT PARAMETERS MEASURED
Biological observations of the exposed mysids and observations of the physical characteristics of the test solutions (e.g., precipitate, film on solution's surface) were recorded at test initiation and at each subsequent 24-hour interval until test termination (96 hours). The criteria used for determining death were the absence of mobility and failure to respond to gentle prodding. Dead organisms were removed and discarded at each observation interval.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0 (dilution water control), 0.10, 1.0, 10, 100 mg a.i./L. (10 mysids per replicate, one replicate per treatment level and the controls). Two exposures were conducted simultaneously in order to define the exposure concentrations for the definitive test and to evaluate the sensitivity of two age classes of mysids. One preliminary exposure was conducted using mysids < 24 hours old. The other preliminary static exposure was conducted using mysids that were 5- to 6-days-old.
- Results used to determine the conditions for the definitive study: Yes. At test termination (96 hours), no mortality or adverse effects were observed among mysids of either age group exposed to any treatment level tested or the control. Since the responses observed for the two age classes of mysids were similar, <24-hour-old mysids were used for the definitive test. Based on these results, nominal concentrations of 13, 22, 36, 60 and 100 mg a.i./L were selected for the definitive exposure.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

At test termination (96 hours), no mortality or adverse effects were observed among mysids exposed to any treatment level tested or the controls.

Analytical Chemistry

Results of the analyses established measured concentrations which were consistent between sampling intervals (i.e., 0 and 96 hours). Mean measured concentrations defined the exposure concentrations as 14, 22, 36, 59 and 100 mg a.i./L. Analysis of the quality control samples resulted in measured concentrations which were consistent with the predetermined recovery range and ranged from 93.5 to 103 % (N = 6) of the nominal fortified levels (10.0 to 100 mg a.i./L). These results established that the appropriate quality control was maintained during the analysis of the exposure solutions.

Water Quality Parameters

The pH decreased with increasing test solution concentration. The remaining water quality parameters measured were unaffected by the concentrations of test material tested and remained within acceptable levels for the survival of mysids.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 96-hour LC50 was determined to be in excess of 100 mg a.i./L.

Executive summary:

The toxicity of the test material to the saltwater invertebrate, Americamysis bahia, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 72-3 and EPA OPPTS 850.1035.

During the study the organisms were exposed to nominal concentrations of test material of 13, 22, 36, 60 and 100 mg a.i./L, the control solution was natural filtered seawater adjusted to a salinity of 20 ± 3 ‰ without the addition of test material or solvent (DMF), and the solvent control was 0.10 mL/L DMF in natural seawater (20 ± 3 ‰). Two replicate vessels were established for each treatment level and the controls. The definitive study was initiated when ten mysids were added to each test vessel (10 per replicate vessel, 20 per treatment level and controls). Biological observations of the exposed mysids and observations of the physical characteristics of the test solutions (e.g., precipitate, film on solution's surface) were recorded at test initiation and at each subsequent 24-hour interval until test termination (96 hours). The criteria used for determining death were the absence of mobility and failure to respond to gentle prodding. Dead organisms were removed and discarded at each observation interval. Live brine shrimp nauplii (Artemia salina) were added to each test vessel containing live test organisms once daily during the exposure period. During the definitive exposure, the pH, dissolved oxygen concentration, temperature, and salinity were measured daily in each test vessel.

At test termination (96 hours), no mortality or adverse effects were observed among mysids exposed to any treatment level tested or the controls. Therefore, under the conditions of the study, the 96-hour LC50 was determined to be in excess of 100 mg a.i./L; the NOEC was 100 mg a.i./L.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June 2001 to 6 November 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 72-2 (Aquatic Invertebrate Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. EPA - FIFRA Standard Evaluation Procedure 540/9-85-005

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

yes

Details on sampling:

SAMPLE COLLECTION/PREPARATION
On day 0 of the study, the concentration of test material in the 100 mg/L bulk dose solution as well as the daphnid dilution water (DDW) control was confirmed by collecting a 3.2 mL aliquot from each test solution and transferring to a 4-mL autosampler vial containing 0.8 mL methanol acidified with 1 % (v/v) acetic acid. On day 2 of the study, samples were taken from test solutions and diluted in the same manner as day 0 samples. This yielded a final sample at 80 % of the initial concentration (dilution correction factor = 1.25) in a matrix of 20/80 acidified methanol/DDW. The resulting solutions were capped and vortexed prior to analysis by HPLC/UV.

METHOD PRECISION
To define method precision, three additional samples were taken on day 0 from the 100 mg test material/L DDW bulk dose solution. These additional samples were collected, diluted and analysed along with the other day 0 samples as described above.

Vehicle:

no

Details on test solutions:

Three replicate vessels (A, B, and C) were prepared at a nominal exposure concentration of 100 mg test material/L on day 0 of the study, in addition to three test vessels of control daphnid dilution water (DDW control). Test solutions were prepared in bulk and then 200-mL aliquots transferred to test vessels immediately before test initiation.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Straus
- Source: in-house culture
- Age at study initiation: Daphnia instars less than 24-hours old from a laboratory-reared culture were used as the test organism
- Feeding during test: No; daphnia were not fed during the test.
- Culture conditions: Rearing conditions were as follows: illumination (cool-white fluorescent) 2050 ± 350 lux; 16-hour light/8-hour dark photoperiod; temperature 20 ± 2 °C. Daphnia were fed a mixed diet of Ankistrodesmus convolutus (algae) and YCT (yeast-ceraphyll trout) chow four times weekly. The day before instars were needed for testing, stock tanks with Daphnia greater than 14-days old which have had at least four broods were removed from the incubator. The instars were separated from adults by gently lifting the screened insert from the 2-L stock tank, releasing instars through the nylon mesh screen while retaining the adult Daphnia. The screened insert, with adult Daphnia, were then placed in another stock tank that contained DDW. 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 Daphnia was put back in place. This procedure was repeated the day the study was set to cull <24-hour old instars for use in the study.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

19.9 - 20.6 °C

pH:

6.2 - 7.6

Dissolved oxygen:

8.6 - 8.7 mg/L (percent oxygen saturation averaged 97 % based on a theoretical value of 8.9 mg/L and remained ≥97 % throughout the exposure).

Nominal and measured concentrations:

0, 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250-mL glass jars containing 200 mL of control or test solution and covered to reduce evaporation
- Aeration: No
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The laboratory water 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. Daphnid water was prepared by adjusting laboratory water to a hardness of about 170 mg/L as CaCO₃ before autoclaving. After adjusting hardness, the water was autoclaved at 250 °C and 18 psi for 30 minutes, cooled, and aerated for approximately 24 hours before use; this water is referred to as daphnid dilution water or DDW.
- Total organic carbon: < 1000 ng/mL
- Chlorine: 5 ppb
- Alkalinity: 36 mg/L CaCO₃
- Hardness: 150 mg/L CaCO₃
- pH: 7.3
- Conductivity: 169 μmho/cm
- Intervals of water quality measurement: Both laboratory and DDW were monitored weekly for pH, alkalinity, conductivity and hardness and twice yearly for total organic carbon (TOC), total suspended solids (TSS), selected inorganics and organic compounds.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light / 8 hours dark
- Light intensity: 1860 - 1970 lux

EFFECT PARAMETERS MEASURED
Daphnia were observed for immobility (inability to swim within 15 seconds after gentle agitation of the test container) at 24 and 48 hours of exposure. Dissolved oxygen, pH, and temperature data were recorded from each bulk dose solution at test initiation (0 hours) and from each individual test vessel (spent test solutions) at 48 hours.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0 (DDW control), 25.0, 50.0, and 100 mg test material/L
- Results used to determine the conditions for the definitive study: Yes. No effects were observed at any of the dose levels following 48 hours of exposure and hence a limit test was conducted at the highest permissible test concentration.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Daphnia were observed at 24 and 48 hours of exposure during this study and the primary endpoint indicating adverse effects was immobility. No effects were observed in the water control or 100 mg test material/L test vessels during this study.

Reported statistics and error estimates:

Based on the design of this study (i.e. limit test), no statistical analysis programs were used for the data analysis and the statistical determination of a NOEC value was not attempted.

Chemical Analysis

Daily residual values were calculated, along with the daily percent of target values (daily value divided by the target value). Averaging the day 0 and day 2 values yielded an overall study average, which was 98.6 % of target. None of the analyses of the dilution water controls exhibited a peak eluting at the retention time of test material at a concentration exceeding the lowest level quantified (LLQ) of 6 mg test material/L-dilution control (concentration of the lowest standard analysed times the dilution factor of 1.25).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 48-hour EC50 was determined to be in excess of 100 mg/L.

Executive summary:

The toxicity of the test material to the freshwater invertebrate, Daphnia magna, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA FIFRA 540 /9-85-005, EPA OPP 72-2, OECD 202 and EU Method C.2.

The study was conducted as a limit test under static conditions with bulk dose solutions prepared on day 0 at a target concentration of 0 (water control) and 100 mg test material/L. Test and control solutions were analysed to determine test material concentrations on days 0 and 2 of the study. Mean measured concentrations were less than the lowest level quantified (LLQ) of 6 mg test material/L for the water controls and 98.6 mg test material/L for the dose solutions. Thirty Daphnia were exposed per dose level in three replicates per concentration with ten Daphnia per replicate. Collected samples were analysed by high performance liquid chromatography with ultraviolet absorbance detection (HPLC/UV). Daphnia were observed at 24 and 48 hours of exposure during this study and the primary endpoint indicating adverse effects was immobility.

No adverse effects were observed in the water control or 100 mg test material/L test vessels during this study. Therefore, under the conditions of the study, the 48-hour EC50 value for the test material with the daphnid, Daphnia magna Straus, was found to be greater than 100 mg/L (equivalent to a mean measured concentration of 98.6 mg test material/L). Due to the study design (i.e. limit test), the statistical calculation of a NOEC was not attempted but was taken to be ≥ 100 mg/L nominal ( ≥ 98.6 mg/L, measured).

Description of key information

Freshwater invertebrates
48 hour EC50 > 100 mg/L (Daphnia magna), EPA FIFRA 540 /9-85-005, EPA OPP 72-2, OECD 202, EU Method C.2, Marino et al. (2001) 
Saltwater invertebrates
96 hour LC50 > 100 mg a.i./L (Americamysis bahia), EPA OPP 72-3, EPA OPPTS 850.1035, Machado (2002) 
96-hour EC50 > 89 mg a.i./L (Crassostrea virginica) EPA OPP 72-3 and EPA OPPTS 850.1025, Cafarella (2002)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Marine water invertebrates

Marine water invertebrates

Effect concentration:

89 mg/L

Additional information

Three studies investigating the short-term toxicity of the substance to aquatic invertebrates are available. All three studies were conducted under GLP conditions and in accordance with standardised guidelines. All three studies were assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997).

In the study reported by Marino et al. (2001) the toxicity of the test material to the freshwater invertebrate, Daphnia magna, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA FIFRA 540 /9-85-005, EPA OPP 72-2, OECD 202 and EU Method C.2.

The study was conducted as a limit test under static conditions with bulk dose solutions prepared on day 0 at a target concentration of 0 (water control) and 100 mg test material/L. Test and control solutions were analysed to determine test material concentrations on days 0 and 2 of the study. Mean measured concentrations were less than the lowest level quantified (LLQ) of 6 mg test material/L for the water controls and 98.6 mg test material/L for the dose solutions. Thirty Daphnia were exposed per dose level in three replicates per concentration with ten Daphnia per replicate. Collected samples were analysed by high performance liquid chromatography with ultraviolet absorbance detection (HPLC/UV). Daphnia were observed at 24 and 48 hours of exposure during this study and the primary endpoint indicating adverse effects was immobility.

No adverse effects were observed in the water control or 100 mg test material/L test vessels during this study. Therefore, under the conditions of the study, the 48-hour EC50 value for the test material with the daphnid, Daphnia magna Straus, was found to be greater than 100 mg/L (equivalent to a mean measured concentration of 98.6 mg test material/L). Due to the study design (i.e. limit test), the statistical calculation of a NOEC was not attempted but was taken to be ≥ 100 mg/L nominal (≥ 98.6 mg/L, measured).

In the study reported by Machado (2002) the toxicity of the test material to the saltwater invertebrate, Americamysis bahia, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 72-3 and EPA OPPTS 850.1035.

During the study the organisms were exposed to nominal concentrations of test material of 13, 22, 36, 60 and 100 mg a.i./L, the control solution was natural filtered seawater adjusted to a salinity of 20 ± 3 ‰ without the addition of test material or solvent (DMF), and the solvent control was 0.10 mL/L DMF in natural seawater (20 ± 3 ‰). Two replicate vessels were established for each treatment level and the controls. The definitive study was initiated when ten mysids were added to each test vessel (10 per replicate vessel, 20 per treatment level and controls). Biological observations of the exposed mysids and observations of the physical characteristics of the test solutions (e.g., precipitate, film on solution's surface) were recorded at test initiation and at each subsequent 24-hour interval until test termination (96 hours). The criteria used for determining death were the absence of mobility and failure to respond to gentle prodding. Dead organisms were removed and discarded at each observation interval. Live brine shrimp nauplii (Artemia salina) were added to each test vessel containing live test organisms once daily during the exposure period. During the definitive exposure, the pH, dissolved oxygen concentration, temperature, and salinity were measured daily in each test vessel.

At test termination (96 hours), no mortality or adverse effects were observed among mysids exposed to any treatment level tested or the controls. Therefore, under the conditions of the study, the 96-hour LC50 was determined to be in excess of 100 mg a.i./L; the NOEC was 100 mg a.i./L.

In the acute toxicity study ino Crassostrea virginica reported by Cafarella (2002), the toxicity of the test material was investigated under GLP conditions and in accordance with the standardised guidelines EPA OPP 72-3 and EPA OPPTS 850.1025.

During the study, oysters were exposed to nominal test concentrations of test material of 13, 22, 36, 60 and 100 mg a.i./L, under flow-through conditions. Dilution water and solvent (DMF) control treatments were included. All treatment levels and the controls were maintained in duplicate. The exposure of oysters was initiated by impartially selecting and placing 20 oysters in each test aquarium (40 per treatment level and the controls). Oysters were spaced equidistant from one another with their valve inflow openings facing toward the flow of water from the Teflon circulator tube. At 24, 48, 72 and 96 hours the oysters were observed for mortality and visible abnormalities. Sub-lethal effects were determined by a comparison of the performance and appearance of the exposed oysters to that of the control oysters. After 96 hours of exposure, the oysters were removed from the test aquaria and the new shell growth was measured microscopically to the nearest 0.1 mm using a calibrated micrometer. During the definitive exposure, the pH, temperature, salinity, and dissolved oxygen concentration were measured daily in each replicate aquarium.

Growth among dilution water control oysters at test termination averaged 2.7 mm, and the growth for the solvent control oysters averaged 2.6 mm. The growth of both control groups during this study was within the historical range of the laboratory. Based on these data, the amount of shell deposition observed during this study is considered representative for this species and acceptable for establishing the relative toxicity of the test material to Eastern oysters. At test termination, no mortality was observed among oysters at any of the treatment levels tested. The percent reduction in shell growth was 12 % for oysters exposed to the highest mean measured concentration tested (89 mg a.i./L). The remaining treatment levels (12, 21, 31 and 50 mg a.i./L) all exhibited a positive response compared to the pooled controls. No sublethal effects were observed among any of the exposed oysters throughout the test concentration range.

Therefore, under the conditions of the study, the 96-hour EC50 was empirically estimated to be greater than 89 mg a.i./L (based on mean measured concentrations). The No-Observed-Effect Concentration (NOEC) was determined to be 89 mg a.i./L.