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

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 October 2010 to 24 November 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This study was conducted for the authorization of Aminopyralid as a PPP active substance, submitted under the directive 91/414 and subsequently authorised under Regulation 1107/2009 where these studies were required to cover the data requirements under this directive and regulation.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage 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:

The concentrations of test material in test solutions were determined in samples collected three days prior to initiation (day -3) of the definitive test and on days 0, 7, 14, 21, 28, 35, and 37 (termination) of the definitive test. A single replicate sample was collected from the control and test material treatments; alternating replicates were sampled at each sampling event (e.g., replicate A sampled on day -3; replicate B sampled on day 0, etc.). The concentrations of test material in the diluter stock solutions were determined in samples collected on the same days as the test solutions.
A 10-mL volume was removed from the control and each test material treatment. Extra control samples were collected to prepare the quality control fortification samples. A 4.5 mL volume of each collected sample was diluted with 0.5 mL acetonitrile (ACN) and further diluted with 90:10 water:ACN to provide final sample concentrations within the range of the analytical standard concentrations (i.e., 80 to 1280 μg/L). A 1.0 mL volume of the diluter stock solution was collected and diluted with 90:10 water:ACN to a concentration with the range of the analytical standard concentrations. Samples were vialed prior to analysis.

Vehicle:

no

Details on test solutions:

Diluter stock solutions were prepared at a target nominal concentration of 156 mg a.i./L at least once every seven days by diluting approximately 2.9714 g (approximately 2.8081 g as active ingredient) or 2.87 L of a 978 mg a.i./L stock solution to a volume of 18 L with dilution water. At each cycle of the diluter system, the FMI pump introduced approximately 40-mL volumes of the diluter stock solution to the diluter system where the solution was diluted with approximately 3910 mL of dilution water, resulting in a final solution volume of approximately 3950 mL.

Test organisms (species):

Cyprinodon variegatus

Details on test organisms:

TEST ORGANISM
- Common name: Sheepshead minnow
- Source: in-house cultures
- Feeding during test: One day following start of hatch (i.e., Study Day 7), fry were fed live brine shrimp (Artemia) nauplii. A standard commercial fish food was added to the diet beginning on study day 23. The fish were fed ad libitum at least three times daily. Food quantity was increased as the test progressed to accommodate the growing fish. The fish were not fed during the 24 hours preceding termination of the definitive test.

METHOD FOR PREPARATION AND COLLECTION OF FERTILISED EGGS
The eggs and milt were collected from mature conditioned sheepshead minnow, which were initially obtained from an in-house culture at the test laboratory. The mature sheepshead minnows were maintained in laboratory saltwater (20 ± 3 ‰) at a temperature of 25 ± 2 °C. Approximately 40 female sheepshead minnow were injected with Human Chorionic Gonadotropin (HCG) solution, containing 1000 International Units (IU) HCG/mL, at a rate of approximately 0.1 mL per fish or approximately 100 IU per fish. Prior to egg collection approximately 8 male sheepshead minnow were sacrificed to obtain testes for fertilisation. Collected testes were maintained in a glass dish containing fresh saltwater until the fertilisation procedure. The injected female sheepshead minnows were anaesthetised with MS-222 and unfertilised eggs were collected by gently stroking the abdomen and subsequently collecting eggs into a glass dish containing fresh saltwater. Following egg collection, the collected testes were placed into a piece of fine mesh netting, macerated, and rinsed with fresh saltwater into the glass dish containing the collected eggs. The milt and eggs were gently swirled and then allowed to remain undisturbed for one to five minutes during fertilisation. The fertilised eggs were then rinsed with fresh saltwater and maintained in a glass dish at approximately 25 °C prior to selection for testing.
A record of biological observations and maintenance activities was kept during the culture period preceding testing. The fish used as sources of eggs/milt did not require any prophylactic or therapeutic disease treatments in the two weeks prior to test initiation.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

37 d

Test temperature:

24.3 - 25.2 °C

pH:

7.9 - 8.8

Dissolved oxygen:

5.9 - 8.8 mg/L

Salinity:

19.2 - 20.6 ‰

Nominal and measured concentrations:

0 (control), 0.10, 0.20, 0.40, 0.80, 1.6 mg a.i./L (nominal)
0 (control), 0.0959, 0.201, 0.401, 0.804, 1.64 mg a.i./L (mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: Test chambers consisted of glass aquaria measuring approximately 15 cm wide by 22 cm long by 24 cm high with a test solution depth of 14 cm. These dimensions yielded a test solution volume of approximately 5 L.
Developing embryos were incubated in glass cups constructed from 9-cm diameter glass jars with Nitex® screen replacing the bottom. The incubation cups were suspended within each replicate aquarium. To facilitate test solution circulation, the cups were oscillated vertically in each aquarium by means of a rocker arm apparatus driven by a low rpm electric motor.
- Type: closed (to stop fish escaping)
- Aeration: No aeration was provided to any control or test material chamber during the test
- Type of flow-through (e.g. peristaltic or proportional diluter): A 2-L proportional equal solvent diluter system, with an FMI metering pump, was used for the intermittent introduction of control and test material solutions into each test chamber during the range-finding and definitive tests. The diluter was constructed from plate glass glued together with silicone adhesive. The diluter system mixing/flow-splitting cells delivered the dilution water control and each of the five test solutions to the test chambers during the definitive test. Each mixing/flow-splitting cell divided each 2-L volume four ways resulting in a volume of approximately 500 mL being delivered to each test chamber with each cycle during the definitive test.
- Renewal rate of test solution (frequency/flow rate): The diluter cycle rate during the definitive test was maintained at approximately 2.6 cycles/hour, which was sufficient to provide approximately 6.2 volume additions to each test chamber over a 24-hour period. Over the course of the definitive test, an average of 31 L per day of control dilution water and test solution was delivered to each chamber.
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was a laboratory saltwater prepared by adding a commercial sea salt mix (Crystal Sea Marinemix; Marine Enterprises International, Inc., Baltimore, Maryland) to laboratory freshwater at a target salinity 20 ± 3 ‰. The laboratory freshwater consisted of well water blended with well water that was demineralised by reverse osmosis to yield water with a total hardness ranging from 130 to 160 mg CaCO₃/L. As the dilution water entered the diluter system, it was heated, aerated, and passed through a particulate filter and an ultraviolet steriliser.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light/ 8-hour dark photoperiod with 30-minute simulated dawn and dusk transition periods.
- Light intensity: 507 to 570 lux

EFFECT PARAMETERS MEASURED
On a daily basis during incubation, the embryos were counted and dead embryos were removed and discarded. Day 0 post-hatch was based on ≥95 % hatch in the control group which occurred on Study Day 9. On study day 15 (i.e., day 6 post-hatch), all live fry were counted and released into their respective replicate growth chamber. Embryos that had not yet hatched by the date of release were maintained in the egg cup until they had hatched, at which time they were released into their respective replicate growth chamber. Survival was monitored daily by visually inspecting each test chamber, and any behavioural or physical changes were recorded, including abnormalities.
After 28 days of post-hatch growth (Study Day 37), surviving fish were carefully netted from each replicate chamber and euthanised with tricaine methanesulfonate. All individuals were measured for standard length (i.e., tip of the snout to the caudal peduncle) using a millimetre scale and blotted wet weight using an electronic balance.
Temperature, pH, salinity and dissolved oxygen concentration were measured in all replicates of the test material treatments and control groups at test initiation, weekly throughout the test, and at termination of the definitive test. Temperature and pH were measured with a WTW pH 330i pH meter or WTW Multi 3500i multimeter. Salinity was measured with a WTW Cond 3300i conductivity/salinity meter or WTW Multi 3500i multimeter. Dissolved oxygen concentration was measured with a WTW OXi 330 dissolved oxygen meter or WTW Multi 3500i multimeter. A continuous recording of temperature in a centrally located test chamber (control replicate C) was made using a datalogger and thermistor probe.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0 (control), 0.65, 1.3, 2.5, 5.0, and 10 mg a.i./L. A total of 20 embryos were added to each incubation cup at initiation of the test. The test chambers were replicated four times per treatment yielding 80 embryos per treatment group.
- Results used to determine the conditions for the definitive study: Yes. Egg hatchability was 85, 93, 93, 88, 91, and 86 % in the 0 (control), 0.65, 1.3, 2.5, 5.0, and 10 mg a.i./L treatments, respectively. All embryos had hatched by study termination, with the exception of one embryo in replicate B of the 2.5 mg a.i./L treatment. Hatch was completed in the control replicates on study day 8 and the day hatch was completed in the test material treatment replicates ranged from study day 10 to study day 18. Fry survival at the end of the 22-day exposure (14 days post-hatch) was 100, 92, 96, 73, 45, and 31 % in the 0 (control), 0.65, 1.3, 2.5, 5.0, and 10 mg a.i./L treatments, respectively. At the termination of the range-finding test, the standard length of the surviving fry from replicates A and B of the control and the 0.65, 1.3, and 2.5 mg a.i./L treatments, as well as replicate C of the 5.0 mg a.i./L treatment and replicate D of the 10 mg a.i./L treatment was measured. The average standard length for fry in the control was 10.1 mm. The percent reduction in average standard length for all test material treatments, as compared to the control, ranged from 8 % at 0.65 mg a.i./L to 60 % at 10 mg a.i./L. All fry measured for standard length were blotted dry, grouped by replicate, and then weighed. The average weight per control fish was 0.0181 g. The percent reduction in average weight for all test material treatments, as compared to the control, ranged from 18 % at 0.65 mg a.i./L to 95 % at 10 mg a.i./L. The results of this range-finding test were used to establish the definitive nominal test concentrations.

Reference substance (positive control):

no

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: time to hatch start, overall hatching success, post-hatch fry survival

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: time to hatch completion

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

length

Remarks on result:

other: Standard length

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

weight

Remarks on result:

other: Blotted Wet Weight

Details on results:

BIOLOGICAL EFFECTS
- Egg hatch: Egg hatch began on day 7 in the control and all test substance treatments. The mean study day hatch started in the 0 (control), 0.10, 0.20, 0.40, 0.80, and 1.6 mg a.i./L was 7, 8, 8, 8, 9, and 8, respectively. There was no statistically significant (p = 0.05) delay in hatch start as compared to the control.
- Day 0 post-hatch: Day 0 post-hatch (i.e., ≥ 95 % hatch in the control treatment) was determined to be study day 9. Hatch was completed in all control treatment replicates between study days 8 and 9. Hatch was completed in all test material treatment replicates between study days 9 and 21. There was a statistically significant delay in time to hatch completion as compared to the control in the 0.20, 0.40, 0.80, and 1.6 mg a.i./L nominal treatments.
- Overall hatching success: Overall hatching success in the control was 94 %, which met the acceptability criterion for this endpoint. Hatching success in the test material treatments ranged from 89 % in the 0.80 mg a.i./L nominal treatment to 96 % in the 0.40 and 1.6 mg a.i./L nominal treatments. There was no statistically significant (p = 0.05) reduction in hatching success as compared to the control.
- Post-hatch survival: Post-hatch survival was calculated as the percent of hatched fry that were alive at test termination (study day 37; 28 days post-hatch). Post-hatch survival in the control was 100 %, which met the acceptability criterion for this endpoint. Post-hatch survival in the test material treatments were 96, 96, 96, 96, and 95 % in the 0.10, 0.20, 0.40, 0.80, and 1.6 mg a.i./L nominal treatments, respectively. There was no statistically significant (p = 0.05) reduction in post-hatch survival observed in any of the test material treatments, as compared to the control.
- Growth: Mean standard length in the control was 14.0 mm. Mean standard length in the test material treatments ranged from 13.2 mm in the 0.80 and 1.6 mg a.i./L nominal treatments to 14.0 mm in the 0.10 mg a.i./L nominal treatment. Mean blotted wet weight in the control was 0.076 g. The relative standard deviation for the blotted wet weight data for the control was 18 %, which was within the acceptability criterion for this endpoint. Mean blotted wet weight in the test material treatments ranged from 0.067 g in the 0.80 and 1.6 mg a.i./L nominal treatments to 0.073 g in the 0.10 mg a.i./L nominal treatment. There were statistically significant (p = 0.05) reductions in length of the 0.40, 0.80, and 1.6 mg a.i./L nominal treatment fry, and in weight of the 0.80 and 1.6 mg a.i./L nominal treatment fry as compared to the control fry.

Analytical Chemistry

The concentrations of test material in test solutions were determined in samples collected three days prior to initiation (day -3) of the definitive test and on days 0, 7, 14, 21, 28, 35, and 37 (termination) of the definitive test. Samples were vialed and analysed using HPLC-UV.

The mean measured test concentrations of test material in the test-material treatments for the 37-day exposure were 0.0959, 0.201, 0.401, 0.804, and 1.64 mg a.i./L and ranged from 96 to 103 % of the nominal concentrations and from 99 to 106 % of the day-0 measured concentrations. All measured concentrations were ± 20 % of the day-0 measured concentrations. No residues of test material above 0.0700 mg a.i./L, the MQL established during the definitive test, were detected in the control. The measured concentration of the diluter stock solutions ranged from 99 to 110 % of the nominal concentrations during the exposure period. Recoveries of test material in the QC fortification samples ranged from 95 to 109 % of the nominal concentrations during the exposure period. Because all measured concentrations were within 20 % of the day-0 measured concentrations, all biological response results were based upon the nominal concentrations of test material for the 37 days of exposure.

Water Quality Parameters

Water quality parameters remained within acceptable testing limits throughout the test. Test solution temperature ranged from 24.3 to 25.2 °C. The continuous temperature recording confirmed the temperature remained within the range of 25 ± 2 °C specified in the protocol. Dissolved oxygen concentrations ranged from 5.9 to 8.8 mg/L (82 to 122 % saturation) in all solutions during the test. Test solution pH values ranged from 7.9 to 8.8 in all solutions during the test. The salinity of all test solutions ranged from 19.2 to 20.6 ‰ during the test.

All control and test material treatments appeared clear and colourless with no visible particulates, surface film, undissolved test material, or precipitate throughout testing.

Validity criteria fulfilled:

yes

Conclusions:

Based on time to hatch start, the NOEC was 1.6 mg a.i./L, based on time to hatch completion the NOEC was 0.10 mg a.i./L, and based on hatching success, and post-hatch survival, the NOEC was 1.6 mg a.i./L. The NOEC for standard length was 0.20 mg a.i./L while the NOEC for blotted wet weight was 0.40 mg a.i./L. There were no morphological or behavioural abnormalities observed during the exposure.

Executive summary:

The chronic toxicity of the test material on sheepshead minnow embryos and fry during an early life-stage exposure was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline EPA OPPTS 850.1400.

The study involved a flow-through exposure system in which embryos and larvae were exposed to nominal concentrations of 0 (control), 0.10, 0.20, 0.40, 0.80, and 1.6 mg a.i./L. The test was initiated when a target number of 20 embryos were distributed to an egg cup in each of four test chambers for the control and each test material treatment, yielding a target number of 80 embryos per treatment group. On a daily basis during incubation, the embryos were counted and dead embryos were removed and discarded. Day 0 post-hatch was based on ≥ 95 % hatch in the control group which occurred Study Day 9. On study day 15 (i.e., day 6 post-hatch), all live fry were counted and released into their respective replicate growth chamber. Embryos that had not yet hatched by the date of release were maintained in the egg cup until they had hatched, at which time they were released into their respective replicate growth chamber. Survival was monitored daily by visually inspecting each test chamber, and any behavioural or physical changes were recorded, including abnormalities. One day following start of hatch (Study Day 7), fry were fed live brine shrimp (Artemia) nauplii. A standard commercial fish food was added to the diet beginning on study day 23. The fish were fed ad libitum at least three times daily. The fish were not fed during the 24 hours preceding termination of the definitive test.

The test chambers were cleaned periodically (at least two times each week following the initial feeding) during the test to remove waste material and uneaten food and to minimise biological growth on the sides and bottom of the test chamber. After 28 days of post-hatch growth (Study Day 37), surviving fish were carefully netted from each replicate chamber and euthanised. All individuals were measured for standard length and blotted wet weight. Temperature, pH, salinity, and dissolved oxygen concentration were measured in all replicates of the test material treatments and control groups at test initiation, weekly throughout the test, and at termination of the definitive test.

There were no morphological or behavioural abnormalities observed during the exposure. Furthermore, there were no effects on time to hatch start, overall hatching success, or post-hatch survival, noted in any of the test material treatments when compared to the control. There was a statistically significant delay in time to hatch completion as compared to the control, at test material treatments at and above 0.2 mg a.i./L. Growth of surviving fry was affected by treatment with the test material; standard lengths were statistically lower in treatments at and above 0.40 mg a.i./L, and on blotted wet weight in treatments at and above 0.80 mg a.i./L.

Therefore, under the conditions of the study, the 32-day NOEC based on: time to hatch start, overall hatching success and post-hatch survival was 1.6 mg a.i./L. Based on time to hatch completion the NOEC was 0.10 mg a.i./L. The NOEC for standard length was 0.20 mg a.i./L while the NOEC for blotted wet weight was 0.40 mg a.i./L.

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 February 2002 to 28 March 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA-FIFRA: Hazard Evaluation Division, Standard Evaluation Procedure: Fish Early Life-Stage: EPA-540/86-138.

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 AND PREPARATION FOR ANALYSIS
Aquaria were sampled on a weekly schedule throughout the study (days 0, 7, 14, 21, 28 and 36). On day 0 (study initiation) individual samples were obtained from each of the 32 test aquaria; a 4-mL aliquot was withdrawn from each aquarium using a disposable glass pipette and transferred to a 4 mL autosampler vial for analysis by HPLC/UV. On day 36 (study termination) individual samples were obtained from the remaining 24 test aquariums and analysed by HPLC/UV. The high two dose levels (8 aquariums, 4 per dose level) were not sampled due to complete mortality.
On each of the sampling days between days 0 and 36, a single replicate aquarium was sampled from each exposure level. Replicate aquariums having the same identifiers (A, B, C, and D) were sampled across all exposure levels for a given sampling date. A non-random, rotational sampling pattern was followed (replicates “A” were sampled on day 7, replicates “B” were sampled on day 14, replicates “C” were sampled on day 21, and replicates “D” were sampled on day 28). Samples were obtained and analysed in the same manner as described in the preceding paragraph.
On each sampling day, the mixing chamber was sampled at the end of a renewal cycle as it was draining; the 4 mL aliquot was transferred to a 4-mL autosampler vial. The dose stocks (spent and fresh where applicable) were also sampled by collecting a 2 mL aliquot which then was diluted 1:10 000 in laboratory dilution water (LDW) and transferred into separate 4 mL autosampler vials. The mixing chamber and dose stock samples were analysed for test material concentration by HPLC/UV with the weekly test vessel samples.

PRECISION
To define method precision, three additional samples were taken on day 0 from replicate “A” of the 0.780-mg/L-exposure level and from replicate “A” of the 10.0 mg/L-exposure level. These additional samples were collected, and analysed along with the other day 0 samples as described above.

Vehicle:

yes

Details on test solutions:

Four replicate aquaria (A, B, C, and D) were maintained at each of the six nominal exposure concentrations in addition to four water (LDW) control aquaria and four solvent control (0.085 mL/L N’,N’-dimethyl formamide (DMF) dosed control water) aquaria. Test vessels were replenished on a regular intermittent cycle with test solutions delivered from a flow-through proportional diluter, which was fortified from a primary feedstock at a nominal concentration of approximately 118 mg test material/mL-DMF. During each cycle of the diluter, 0.5 mL of the feedstock was added the mixing chamber filled with 5.91 L of LDW, yielding a nominal concentration of 10.0 mg test material/L-LDW. This solution (undiluted becomes top dose level) was then pumped into the cascading dilutor vessels which resulted in five, 60 percent serial dilutions. The test solutions were then dumped from the cascading dilutors into splitter cells, which equally dispersed the dose solutions into the four replicate test vessels at each dose level. Fresh diluter feedstocks were prepared on a weekly basis throughout the study.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Feeding during test: Test fish within two days post-hatch (within 2 days following 90 % hatching of the controls) were fed a diet of newly hatched brine shrimp (Artemia sp.) twice daily with at least six hours between feedings. The concentrated brine shrimp were added to each test vessel in equal amounts varying from 200 µL per feeding at the beginning of the study up to 475 µL per feeding near the end of the study. Green algae (Selenastrum capricornutum) was used to supplement the diet, at least once daily (0.5 mL), starting the day following first hatch to 4 days following the initiation of brine shrimp feeding. Fish were not fed during the 24 hours preceding test termination.

ACCLIMATION
- Acclimation period: Upon arrival, the embryos were visually inspected and acclimated to laboratory conditions at a temperature of 25 ± 2 °C. Prior to test initiation, all embryos were pooled in a large glass dish. The embryos were visually inspected using a stereo microscope and those that appeared abnormal or fungus-infected were discarded. Only normal appearing embryos were used to start the test. Post fertilised, non-eyed embryos were used to initiate the study and were approximately 20-26 hours old following microscopic observations.
- Acclimation conditions: same as test

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

36 d

Hardness:

53 - 73 mg CaCO₃/L

Test temperature:

24.5 - 25.5 °C

pH:

6.9 - 8.2

Dissolved oxygen:

6.3 - 9.9 mg/L

Nominal and measured concentrations:

0 (laboratory water), 0 (solvent control of 0.085 mL/L DMF dosed control water), 0.780, 1.30, 2.16, 3.60, 6.00 and 10.0 mg test material/L (nominal)
0 (laboratory water), 0 (solvent control of 0.085 mL/L DMF dosed control water), 0.708, 1.36, 2.44, 3.89, 6.71 and 11.4 mg test material/L (mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: Test aquaria were constructed of double-strength glass held together with clear silicone adhesive and measured approximately 15 x 10 x 9 cm. Each was provided with a cover and a Nitex® (Tetko, Elmsford, New York) screen-covered drain guard that maintained a test volume of approximately 850 mL.
- Embryo cups: Embryos were incubated in circular incubation cups (7.5-cm high x 7-cm diameter) that were suspended in a cylindrical glass test chamber (8.5-cm high x 8.5-cm diameter). Both vessel types contained ~360 µm screen-covered bottoms and were supported by 1-cm glass beads attached to screen bottoms. Flow from the delivery tubes was directed in and around the incubation cups to produce an intermittent flow of test solution around the embryos during the incubation period. The incubation cups were removed on exposure day 12 of the study.
- Aeration: No data
- Type of flow-through: Intermittent-flow proportional diluter. The system used was designed to provide up to six test concentrations, a solvent control, and a laboratory water control. The diluter was calibrated so that the concentration of the test material in each treatment was approximately 60 % of that in the next higher treatment level. The diluter operates as follows: a precision dosing system (Hamilton MICROLAB 500 system) delivers a designated amount of the concentrated test material stock solution from a glass carboy to the mixing chamber where it is mixed with water and then distributed to toxicant cells. The mixing chamber is equipped with a recirculating pump that facilitates dissolution and mixing of the test material. When the diluter cycles, the test material from each toxicant cell blends with water from its respective water cell and flows into mixing/splitting chambers. Glass delivery tubes from these chambers provide approximately 500 mL of test solution to each of four replicate test aquaria.
- Renewal rate of test solution (frequency/flow rate): During the test, the diluter system provided an average of 9.4 volume turnovers in each test vessel during each 24-hour period.
- No. of fertilised eggs/embryos per vessel: 25
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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 near Whitestone Point and was limed and flocculated with ferric chloride. The water was pumped to the laboratory prior to municipal treatment. Before use in the laboratory, the water was sand-filtered, pH-adjusted with gaseous CO₂, carbon-filtered, and UV-irradiated.
- Total organic carbon: <1000 ng/mL
- Chlorine: <1 to 7 ppb
- Alkalinity: 30 - 43 mg/L CaCO₃
- Conductivity: 155 - 207 µmhos/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: The alkalinity, conductivity, pH, and hardness of the laboratory water are monitored weekly. Total organic carbon, total suspended solids, and selected inorganic and organic compounds are monitored biannually.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours of light / 8 hours of darkness
- Light intensity: 660 - 714 lux

EFFECT PARAMETERS MEASURED
The embryos/larvae were observed and counted daily in each test vessel until hatching was complete (up to day 5); dead or fungus-infected embryos/larvae were removed at each observation. Upon completion of hatching, the percent of embryos that hatched and the day-to-mean hatch for each replicate were recorded.
Larvae were observed for mortality at least once daily and recorded. Larvae were observed at least once weekly and at test termination with mortality and sublethal effects recorded. Dead larvae were removed when recorded. The test was conducted for 32 days post hatch (when hatching is 90 %) of the controls (day 4) for a total of 36 days at which time larvae survival, overall survival and the percent of normal larvae was calculated for each replicate. At test termination, all surviving fish were sacrificed for wet (blotted dry) weight (nearest 1 mg) and standard length (nearest 1 mm) measurements. All fish were euthanised with tricaine methanesulfonate prior to length and weight measurements.
Dissolved oxygen, pH and temperature data were recorded on test days 0, 7, 14, 21, 28, and 36 (test termination) in each test and control vessel with surviving organisms. Water temperature was maintained at 25 ± 2 °C and did not differ by more than ± 1.5 °C between test chambers or between successive days at any time during the test. Water temperature was also continuously monitored from one test vessel throughout the study. Loading did not exceed 0.5-g fish per litre of test solution passing through the test vessels in 24 hours, and did not exceed 5 g/L of test solution at any time. The dissolved oxygen levels were greater than 75 % of saturation throughout the exposure. Water quality parameters such as alkalinity, hardness, pH and conductivity were measured on test days 0, 7, 14, 21, 28, and 36 (test termination) from a control group and one exposure group. The water sample was collected via the splitter cells for each of the respective groups.

VEHICLE CONTROL PERFORMED: yes

Reference substance (positive control):

no

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

1.36 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: weight, length, percent normal larvae at termination, percent larvae survival, percent overall survival

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

> 11.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: time to hatch, number hatched

Details on results:

Abnormal larvae observations were recorded during the study and were sporadic across all dose levels. Abnormal observations recorded included pale colouration, immobility, deformed/under-developed body, and scoliosis.
Based on the measured weights of the larvae measured at the end of the study and a test vessel volume of 850 mL with an average of 9.4 turnovers a day, the biological loading did not exceed 0.5-g fish per litre of test solution passing through the test vessels in 24 hours, and did not exceed 5 g/L of test solution at any time. Wet (blotted dry) weights were conducted on all surviving larvae at test termination. The calculated wet weight measurements indicated the maximum loading within the test vessels to be 0.113 mg fish/ L/day and 1.067 g-fish/L.

Reported statistics and error estimates:

The NOEC, LOEC, and MATC values were estimated using mean analysed concentrations for % overall survival, % larvae survival, the % of embryos hatched, the % normal larvae at test termination, days-to-mean hatch and the growth parameters (weight and length) at the end of the test. The day-to-mean hatch and growth (length and weight) data were first tested for normality using the Shapiro- Wilk’s test at a type I error rate of 0.01. If the data was not normally distributed, the logarithmic inverse and square root transformations were tested sequentially to search for a normalising transformation. Next, the data and transformed variables were tested for homogeneity of variance using Bartlett’s test at a type I error rate of 0.01. If the raw data, or a transformed variable, were both normal and homogeneous, a parametric analysis was conducted using a one-tailed Dunnett’s test (for growth) and a two-tailed Dunnett’s test (for day-to-mean hatch) to compare each treated group with the control(s) at a type I error rate of 0.05.
Data that were not normally distributed and/or not homogeneous were analysed parametrically with a Steel’s Many-one rank test if the number of replicates in each treatment group was the same; or with a Kruskal-Wallis test if the number of replicates was different. Steel’s Many-one rank test is one-sided and the Kruskal-Wallis test is two-sided, both with a type I error rate of 0.05. A significant result in the Kruskal-Wallis test leads to a pairwise comparison of each treatment with the control(s) using the Wilcoxon procedure at a type I error rate of 0.01 (one-sided). Both the Steel’s test and the Wilcoxon test lead to the determination of a NOEC. The % of embryos that hatched, % normal larvae at test termination, % larvae that survived and % overall survival parameters were arcsine square root transformed and analysed by a parametric analysis of variance followed by a one-tailed Dunnett’s test at a type I error rate of 0.05.

Chemical Analysis

A study average and percent of target were calculated for each dose level by averaging the six daily values. Study average percent recovery of target dose levels for test material ranged from 90.5 to 114 %, indicating the solutions were delivered to the aquaria at their intended concentrations. The overall average percent of target concentration and standard deviation values (for the entire study, n = 6) were 107 ± 8.80 %. The weekly analyses of the mixing chamber and dose stocks were in good correlation with the target concentrations. The mixing chamber concentration ranged from 10.3 to 11.6 mg test material/L-laboratory dilution water (10.0 mg/L target concentration).

None of the analyses of the water controls or DMF controls exhibited a peak eluting at the retention time of the test material at a concentration exceeding the lowest level quantified of 0.09 mg test material/L-laboratory dilution water (approximate concentration of the lowest standard analysed).

Water Quality Parameters

The parameters measured to verify the water quality were: hardness expressed as mg CaCO₃/L, alkalinity expressed as mg CaCO₃/L, conductivity expressed as µmhos/cm and residual chlorine expressed as parts per billion (ppb). The hardness throughout the study ranged from 53 to 73 mg CaCO₃/L and alkalinity ranged from 30 to 43 mg CaCO₃/L. Residual chlorine throughout the study ranged from < 1 to 7 ppb and conductivity ranged from 58.7 to 68.7 µmhos/cm. Light measurements were recorded weekly from the middle of the diluter and ranged from 660 to 714 lux.

The pH throughout the study averaged 7.4 and ranged from 6.9 to 8.2. Test temperature during the study averaged 24.9 °C and ranged from 24.5 to 25.5 °C. Dissolved oxygen (DO), expressed in mg/L, ranged from 6.3 to 9.9 and averaged 8.6 mg/L during the study. Dissolved oxygen (DO), expressed as percent air saturation, ranged from 78 to 122 % and averaged 106 % during the study.

 

Table 1: Summary of Statistical Findings on Measured Effect Endpoints

Parameter	NOEC (mg/L)	LOEC (mg/L)	MATC (mg/L)
Weight (mg)	1.36	2.44	1.82
Length (mm)	1.36	2.44	1.82
Percent Embryos Hatched	>11.4	>11.4	>11.4
Days-to-Mean Hatch	>11.4	>11.4	>11.4
Percent Normal Larvae at Test Termination	1.36	2.44	1.82
Percent Larvae Survival	1.36	2.44	1.82
Percent overall Survival	1.36	2.44	1.82

Values expressed as mean analysed test material concentration

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the NOEC was determined to be 1.36 mg/L.

Executive summary:

The chronic toxicity of the test material was investigated to the early life-stages of the fathead minnow. The study was conducted under GLP conditions and in accordance with the standardised guidelines EPA-540/86-138 and OECD 210.

The study involved a flow-through exposure system in which embryos and larvae were exposed to nominal concentrations of 0 (water control), 0 (solvent control of 0.085 mL/L DMF dosed control water), 0.780, 1.30, 2.16, 3.60, 6.00, and 10.0 mg test material/L, which resulted in mean measured concentrations of less than the lowest level quantified of 0.09 mg test material/L for the water and solvent controls, 0.706, 1.36, 2.44, 3.89, 6.71, and 11.4 mg test material/L, respectively. Test aquaria were sampled on a weekly schedule throughout the duration of the study and the samples analysed for test material using high performance liquid chromatography with ultraviolet detection (HPLC/UV). One hundred fathead minnow embryos (four replicates of 25 embryos per dose level) approximately 20- to 26-hours post fertilisation were used to initiate the test. The test system was maintained for 32 days post hatch of the control embryos (36 days total). The effect endpoints evaluated were the number of embryos that hatched (embryo survival), time to hatch (day-to-mean hatch), normal larvae at test termination, larvae survival, overall survival, and growth (length and weight).

No statistically significant (alpha = 0.05) effects were observed in this study up through the highest exposure level tested of 11.4 mg test material/L for the percent embryos hatched and days-to-mean hatch endpoints. Statistically significant (alpha = 0.05) effects were observed in all remaining endpoints evaluated down to the 2.44 mg test material/L exposure level. Therefore, under the conditions of the test, the NOEC, LOEC, and MATC for this study were determined to be 1.36, 2.44, and 1.82 mg test material/L, respectively.

Description of key information

Freshwater fish
36 day NOEC (weight, length, percent normal larvae at termination, percent larvae survival, percent overall survival) = 1.36 mg/L (fathead minnow), EPA-540/86-138, OECD 210, Marino et al. (2003) 
Saltwater fish
37 day NOEC (time to hatch start, overall hatching success, post-hatch survival) = 1.6 mg a.i./L, 37 day NOEC (time to hatch completion) = 0.10 mg a.i./L, 37 day NOEC (standard length) = 0.20 mg a.i./L, 37 day NOEC (blotted wet weight) = 0.40 mg a.i./L (sheepshead minnow), EPA OPPTS 850.1400, Hicks (2011)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.36 mg/L

Marine water fish

Marine water fish

Effect concentration:

0.1 mg/L

Additional information

Two studies, investigating the long-term toxicity of the substance to fish are available. Both studies were conducted under GLP conditions and in accordance with standardised guidelines. Both 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. (2003) the chronic toxicity of the test material was investigated to the early life-stages of the fathead minnow. The study was conducted under GLP conditions and in accordance with the standardised guidelines EPA-540/86-138 and OECD 210.

The study involved a flow-through exposure system in which embryos and larvae were exposed to nominal concentrations of 0 (water control), 0 (solvent control of 0.085 mL/L DMF dosed control water), 0.780, 1.30, 2.16, 3.60, 6.00, and 10.0 mg test material/L, which resulted in mean measured concentrations of less than the lowest level quantified of 0.09 mg test material/L for the water and solvent controls, 0.706, 1.36, 2.44, 3.89, 6.71, and 11.4 mg test material/L, respectively. Test aquaria were sampled on a weekly schedule throughout the duration of the study and the samples analysed for test material using high performance liquid chromatography with ultraviolet detection (HPLC/UV). One hundred fathead minnow embryos (four replicates of 25 embryos per dose level) approximately 20- to 26-hours post fertilisation were used to initiate the test. The test system was maintained for 32 days post hatch of the control embryos (36 days total). The effect endpoints evaluated were the number of embryos that hatched (embryo survival), time to hatch (day-to-mean hatch), normal larvae at test termination, larvae survival, overall survival, and growth (length and weight).

No statistically significant (alpha = 0.05) effects were observed in this study up through the highest exposure level tested of 11.4 mg test material/L for the percent embryos hatched and days-to-mean hatch endpoints. Statistically significant (alpha = 0.05) effects were observed in all remaining endpoints evaluated down to the 2.44 mg test material/L exposure level. Therefore, under the conditions of the test, the NOEC, LOEC, and MATC for this study were determined to be 1.36, 2.44, and 1.82 mg test material/L, respectively.

 

In the study reported by Hicks (2011) the chronic toxicity of the test material on sheepshead minnow embryos and fry during an early life-stage exposure was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline EPA OPPTS 850.1400.

The study involved a flow-through exposure system in which embryos and larvae were exposed to nominal concentrations of 0 (control), 0.10, 0.20, 0.40, 0.80, and 1.6 mg a.i./L. The test was initiated when a target number of 20 embryos were distributed to an egg cup in each of four test chambers for the control and each test material treatment, yielding a target number of 80 embryos per treatment group. On a daily basis during incubation, the embryos were counted and dead embryos were removed and discarded. Day 0 post-hatch was based on ≥ 95 % hatch in the control group which occurred Study Day 9. On study day 15 (i.e., day 6 post-hatch), all live fry were counted and released into their respective replicate growth chamber. Embryos that had not yet hatched by the date of release were maintained in the egg cup until they had hatched, at which time they were released into their respective replicate growth chamber. Survival was monitored daily by visually inspecting each test chamber, and any behavioural or physical changes were recorded, including abnormalities. One day following start of hatch (Study Day 7), fry were fed live brine shrimp (Artemia) nauplii. A standard commercial fish food was added to the diet beginning on study day 23. The fish were fed ad libitum at least three times daily. The fish were not fed during the 24 hours preceding termination of the definitive test.

The test chambers were cleaned periodically (at least two times each week following the initial feeding) during the test to remove waste material and uneaten food and to minimise biological growth on the sides and bottom of the test chamber. After 28 days of post-hatch growth (Study Day 37), surviving fish were carefully netted from each replicate chamber and euthanised. All individuals were measured for standard length and blotted wet weight. Temperature, pH, salinity, and dissolved oxygen concentration were measured in all replicates of the test material treatments and control groups at test initiation, weekly throughout the test, and at termination of the definitive test.

There were no morphological or behavioural abnormalities observed during the exposure. Furthermore, there were no effects on time to hatch start, overall hatching success, or post-hatch survival, noted in any of the test material treatments when compared to the control. There was a statistically significant delay in time to hatch completion as compared to the control, at test material treatments at and above 0.2 mg a.i./L. Growth of surviving fry was affected by treatment with the test material; standard lengths were statistically lower in treatments at and above 0.40 mg a.i./L, and on blotted wet weight in treatments at and above 0.80 mg a.i./L.

Therefore, under the conditions of the study, the 32-day NOEC based on: time to hatch start, overall hatching success and post-hatch survival was 1.6 mg a.i./L. Based on time to hatch completion the NOEC was 0.10 mg a.i./L. The NOEC for standard length was 0.20 mg a.i./L while the NOEC for blotted wet weight was 0.40 mg a.i./L.