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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 August 2002 to 4 September 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 72-4 (Fish Early Life-Stage and Aquatic Invertebrate Life-Cycle Studies)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

yes

Details on sampling:

Aliquots were collected from the bulk dose solutions and individual test vessels at least once per week throughout the course of the study in order to determine the concentration of test material in the solutions. All bulk dose solutions (nominal concentrations of 0 (control), 3.13, 6.25, 12.5, 25.0, 50.0 and 100 mg/L) were sampled and analysed on days 0, 2, 12 and 19. The individual spent test solutions (10 replicates per dose level) were sampled on days 2, 5, 14 and 21. All test solutions were prepared for analysis by collecting 1-mL aliquots and transferring them to individual autosampler vials containing 1 µL phosphoric acid. The samples were mixed and analysed by HPLC/UV.
To assess analytical method precision and test solution homogeneity, three additional aliquots were collected on day 0 from the 3.13 and 100 mg test material/L bulk dose solutions. These additional samples were collected, acidified, and analysed along with the other day 0 samples as described above.

Vehicle:

no

Details on test solutions:

Test solutions for the definitive study were prepared on day -1 at nominal concentrations of 0 (water control), 3.13, 6.25, 12.5, 25.0, 50.0, and 100 mg test material/L. Ten litres of solution for each dose level were prepared on day -1 for use throughout the study. An additional 325 mL of the 6.25 mg/L test solution and 500 mL of the 50.0 mg/L test solution were prepared on exposure day 19 and needed to complete the study. Daphnids were introduced into test vessels containing fresh dose solutions on day 0 and transferred into vessels containing fresh dose solution each Monday, Wednesday, and Friday throughout the study.
The bulk test solutions were prepared by gravimetrically measuring the appropriate amount of test material (adjusting for test material purity) into 20-L glass carboys and filling each carboy with 10 L of daphnia dilution water. Test solutions were stirred and sonicated for approximately two minutes for complete dissolution of material. During the study, the bulk solutions (carboys) were held in the same incubator in which the test vessels were maintained (Lab-Line Instruments, Inc., Dubuque, Iowa, Environmental Chamber EC2211M). Prior to dispensing ~ 90 mL of bulk solution into test vessels (10 replicate vessels per dose level), 1 L of each bulk test solution was transferred into a 1-L volumetric flask. To each flask, ten millilitres of Selenastrum capricornutum suspension (3 x 10⁷ cells/mL) and 5 mL of YCT suspension (2010 mg total solids/L) was added as a daphnid food source. On non-renewal days, 0.5 mL of Selenastrum capricornutum suspension was added to each test vessel.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
Daphnia magna neonates from in-house cultures were used as test organisms. Mass cultures of Daphnia magna were reared in incubators under cool-white fluorescent lights (approximately 2050 ± 350 lux) and a 16-hour light:8-hour dark photoperiod at 20 ± 2 °C.
Daphnids were fed a mixed diet of Selenastrum capricornutum (a green alga) and YCT (yeast, Cerophyll and trout chow suspension) five times per week. Young Daphnia magna were removed from mass cultures the day before test initiation (day -1) by sieving with a 300-µm mesh screen that retains mature individuals while allowing immature daphnids to pass through the mesh. Cultures were sieved again on day 0 and neonates < 24-hours old were collected for exposure testing.

- Feeding during test: Feeding was performed at test solution renewal by adding 10 mL of Selenastrum capricornutum (217 mg organic carbon/L) and 5 mL of YCT (2010 mg total solids/L) to each 1-L test solution flask. Feeding was performed on non-renewal days by adding 0.5 mL of the Selenastrum capricornutum suspension to each test vessel.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

154-273 mg/L as CaCO₃

Test temperature:

19.7-21.1 °C

pH:

6.3-8.7

Dissolved oxygen:

2.9 -10.8 mg/L (average of 90 % saturation over the 21-day exposure period)

Nominal and measured concentrations:

0 (water control), 3.13, 6.25, 12.5, 25.0, 50.0 and 100 mg/L (nominal)
< LLQ, 2.99, 6.16, 12.5, 25.5, 49.8 and 102 mg/L (mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: The test was conducted in 120-mL borosilicate jars containing approximately 90 mL of solution. Vessels were covered with a sheet of Plexiglas® to reduce evaporation.
- Aeration: no
- Renewal rate of test solution: Test solution renewals were performed every Monday, Wednesday, and Friday.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

LABORATORY WATER AND DAPHNID DILUTION WATER
The laboratory water was Lake Huron water supplied to the test site 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 carbon dioxide, carbon-filtered and UV-irradiated. Daphnid dilution water was prepared by adjusting this laboratory water to a hardness of approximately 170 mg/L as CaCO₃. After adjusting hardness, the water was autoclaved at 250 °F and 18 psi for 30 minutes and cooled before use. Both laboratory and daphnid dilution water were monitored weekly for pH, alkalinity, conductivity, and hardness and twice yearly for total organic carbon (TOC), total suspended solids (TSS), and selected inorganic and organic compounds.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours of light / 8 hours of darkness
- Light intensity: 622-925 lux (57.8-85.9 footcandles)

EFFECT PARAMETERS MEASURED
Observations were made and the number of surviving daphnids recorded daily. Mortality was defined as an inability to swim within 15 seconds after gentle agitation of the test container. Reproduction was evaluated by counting surviving and dead Daphnia magna neonates in each test vessel every renewal day (Monday, Wednesday, or Friday) and at test termination. This was performed at the same time as test solution renewal by first transferring the adult daphnid into a new test vessel, followed by sieving the spent solution from the old test vessel. On the final day of the study (day 21), surviving adult daphnids were counted and then individually measured for the growth endpoint (body length excluding the anal spine) using a stereomicroscope.

ENVIRONMENTAL PARAMETERS
Dissolved oxygen, temperature and pH were measured in freshly prepared bulk solutions and all their respective spent solution replicates at least one time per week during the study. Alkalinity, hardness and conductivity were measured at least one time per week in fresh bulk test solutions for the water control and the greatest test material concentrations with surviving organisms (100 mg/L). Pooled spent solutions from the same treatments were evaluated for alkalinity, hardness and conductivity on the following solution renewal day.

RANGE-FINDING STUDY
A 21-day range-finding probe was conducted with eight daphnids (one daphnia/replicate with eight replicates/treatment) exposed to nominal test concentrations of 0.185, 0.410, 0.911, 2.02, 4.50, and 10.0 mg test material/L.
Mortality in the control and all treatment levels was ≤ 20 %, with the exception of the 4.50 mg/L treatment, in which six of eight daphnids (75 %) were dead by the end of the 21-day study. Such anomalous mortality at an intermediate-dose level is not characteristic of a typical dose response, and is not believed to be an effect of the test material. Therefore, interpretation of these probe results indicated that the EC50 for reproduction and survival are both greater than 10.0 mg/L. However, since the mortality at the 4.50 mg/L dose level is unexplained, this dose level was encompassed in the range of dose levels set for the definitive study. No significant effects in reproduction were observed at any the dose levels based on the average number of progeny per surviving first generation (parent) daphnid.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: survival, reproduction, and growth

Details on results:

Survival was 100 % during the test in the control and in all treatment levels.
The mean number of young produced per surviving adult was 150.6 in the water control, 155.1 in the 3.13 mg test material/L, 151.2 in the 6.25 mg test material/L, 166.3 in the 12.5 mg test material/L, 168.8 in the 25.0 mg test material/L, 185.0 in the 50.0 mg test material/L, and 184.7 in the 100 mg test material/L test concentrations. No mortality of offspring was observed during the study.
The mean length per surviving adult was 4.24 mm in the water control, 4.22 mm in the 3.13 mg test material/L, 4.21 mm in the 6.25 mg test material/L, 4.20 mm in the 12.5 mg test material/L, 4.17 mm in the 25.0 mg test material/L, 4.24 mm in the 50.0 mg test material/L, and 4.20 mm in the 100 mg test material/L test concentrations.

Reported statistics and error estimates:

No daphnid mortality was observed during the conduct of this 21-day study, therefore, the statistical evaluation of an LC50 value (mortality endpoint) was not performed. The statistical evaluation of an EC50 value (reproduction endpoint) was not performed, since reduction in reproduction greater than 50 % when compared to the controls was not observed at any of the dose level tested.
The NOEC and LOEC values for reproduction and growth (length) were evaluated by the appropriate statistical procedures. The raw data were first tested for normality using the Shapiro-Wilk's test at a Type I error rate of 0.01. If the data were not normally distributed, the logarithmic, inverse and square root transformations were tested sequentially to search for a normalising transformation. Next, the data and the same 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 Dunnett's test to compare each treated group with the control. A one-tailed Dunnett's test, looking for a significant decrease from the control group, was conducted at a Type I error rate of 0.05.
Data that were not normally distributed and/or not homogeneous were analysed non-parametrically with a Steel's Many-One Rank Test if the number of replicates in each treatment group were the same. A Kruskal-Wallis test was utilised 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 have 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 using the Wilcoxon procedure having 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.

Chemical Analysis

Mean analysed concentrations of test material were: less than the lowest level quantified of 0.251 mg test material/L for the control group, and 2.99, 6.16, 12.5, 25.5, 49.8, and 102 mg test material/L for the treated solutions, respectively. The overall average percent of target and standard deviation values for the entire study were 99.7 ± 2.43 %.

None of the analyses of the controls exhibited a peak eluting at the retention time of test material at a concentration exceeding the lowest level quantified of 0.251 mg test material/L, which was the concentration of the analyte in the lowest standard analysed.

Water Quality Measurements

Test solution dissolved oxygen levels ranged from 2.9 - 10.8 mg/L (8.0 ± 2.0 mg/L) and averaged 90 % of saturation over the 21-day exposure period. The lowest dissolved oxygen measurement of 2.9 mg/L was recorded on exposure day 14 from two spent control solutions. These were the only vessels during the conduct of this study to decline below the OECD Method 211 test guideline suggested criterion of 3 mg/L; no effects on the test organisms were observed in these replicate control vessels during this study. Test solution temperatures ranged from 19.7 - 21.1 °C (20.3 ± 0.3 °C) and pH ranged from 6.3 - 8.7 (7.6 ± 0.7) during the study. The hardness of the test solution water during the test ranged from 154 - 273 mg/L as CaCO₃. Alkalinity measurements ranged from 18-22.5 mg/L as CaCO₃, with the exception of one measurement of 55.8 mg/L, from which the sample had been spilled and only a small sample volume remained available for measuring; this may have added error to the measurement. The conductivity of the test solutions ranged from 307.3 - 519.9 μmhos/cm. Illumination (16-hour light/8-hour dark photoperiod) during the study ranged from 622 - 925 lux (57.8 - 85.9 footcandles), with an average of 57.8 - 85.9 footcandles (74.7 ± 7.5 footcandles).

Table 1: Summary of Biological Data

Nominal conc. (mg/L)	Mean measured conc. (mg/L)	Percent survival	Average progeny per surviving adult (mean ± SD)	Length in mm (mean ± SD)
0 (control)	0 (<LLQ)	100	150.6 ± 21.1	4.24 ± 0.07
3.13	2.99	100	155.1 ± 43.1	4.22 ± 0.05
6.25	6.16	100	151.2 ± 34.3	4.21 ± 0.08
12.5	12.5	100	166.3 ± 32.0	4.20 ± 0.10
25.0	25.5	100	168.8 ± 18.8	4.17 ± 0.03
50.0	49.8	100	185.0 ± 24.3	4.24 ± 0.12
100	102	100	184.7 ± 19.7	4.20 ± 0.05

LLQ = Lowest level quantified, 0.251 mg test material/L

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 21-day NOEC for survival, reproduction and growth, was found to be 100 mg/L.

Executive summary:

The chronic toxicity of the test material to the freshwater invertebrate, Daphnia magna Straus, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 211 and EPA OPP 72-4.

During the study, the Daphnia were exposed to concentrations of test material of 0 (water control), 3.13, 6.25, 12.5, 25.0, 50.0, and 100 mg/L. Test solutions were renewed every Monday, Wednesday, and Friday during the course of this study. Each test material treatment and the water control group were set using 10 replicates, each containing a single daphnid. Endpoints of interest in this study included survival, reproduction (total progeny/surviving adult) and growth (length). Feeding, as described above under Test Solutions, was performed at test solution renewal by adding 10 mL of Selenastrum capricornutum (217 mg organic carbon/L) and 5 mL of YCT (2010 mg total solids/L) to each 1-L test solution flask. Feeding was performed on non-renewal days by adding 0.5 mL of the Selenastrum capricornutum suspension to each test vessel. Test vessels were maintained in an incubator at 20 ± 1 °C during the exposure. Study initiation and transfers of test organisms occurred immediately following sampling of bulk test solutions for analytical confirmation.

The test solutions resulted in mean analysed concentrations of less than the lowest level quantified of 0.251 mg test material/L for the control group, and 2.99, 6.16, 12.5, 25.5, 49.8, and 102 mg test material/L for the treated solutions, respectively. The overall average percent of target and standard deviation values for the entire study were 99.7 ± 2.43%.

Survival was 100 % in the control and in all treatment levels. The statistically derived NOEC for survival was thus 100 mg/L (equivalent to 102 mg/L, mean measured), the highest concentration tested. No statistically significant differences in reproductive output, determined using mean progeny per surviving adult, were observed in the treatments when compared to the control. The statistically derived NOEC for reproduction was 100 mg/L (equivalent to 102 mg/L, mean measured), the highest concentration tested. The coefficient of variation for control reproduction was 14.0 %. The statistically derived NOEC for growth (length) was 100 mg/L (equivalent to 102 mg/L, mean measured), the highest concentration tested.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 September 2010 to 13 October 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1350 (Mysid Chronic Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 72-4 (Fish Early Life-Stage and Aquatic Invertebrate Life-Cycle Studies)

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 two days prior to initiation (day -2) of the definitive test and on days 0, 8, 14, 21, and 28 (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 –2; 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 from 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 and analysed using HPLC-UV.

Vehicle:

no

Details on test solutions:

A proportional diluter system equipped with a stock solution metering device was used for the preparation of test solutions and intermittent introduction of the solutions to the test chambers.
An LMI metering pump introduced 400 mL volumes of the diluter stock solution to the diluter system, where the diluter stock solution volume was diluted with approximately 3500 mL of dilution water. Diluter stock solutions were prepared at a target concentration of 983 mg a.i./L at least once every six days by diluting approximately 1.04 g (i.e., 0.983 g as active ingredient) of test material per litre of dilution water. The volumes of diluter stock solution prepared prior to test initiation and during the in-life phase ranged from 18 to 200 L.

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
Mysids were obtained from in-house cultures that were maintained at the test laboratory. The adult mysids used to generate the juvenile test organisms were cultured in artificial saltwater with salinity of approximately 20 ‰ and at a temperature of approximately 25 °C. The animals were fed brine shrimp nauplii (Artemia sp. <48 hours old). Juvenile mysids were <24 hours old at test initiation.

- Feeding during test: Mysids were fed ad libitum brine shrimp nauplii (Artemia sp.; 24-48 hours old) at least three times daily except for initiation and termination, when mysids were fed twice. The food stock was enriched the day of use. The enrichment mixture was then added to the food stock at the rate of 50 mL/L of food stock.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

28 d

Test temperature:

24.2 - 25.0 °C

pH:

7.72 - 8.08

Dissolved oxygen:

6.46 - 7.70 mg/L (90 - 97 % saturation)

Salinity:

19.2 to 20.5 ‰

Nominal and measured concentrations:

0 (control), 6.5, 13, 25, 50, 100 mg a.i./L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: The test chambers consisted of all-glass aquaria. Each test chamber used for the definitive test measured approximately 22 cm wide, 77 cm long (maximum length) or 38 cm long (minimum length) and 30 cm high. Each definitive-test chamber had a glass pane in the middle of the tank containing two holes near the bottom of the tank. This partition effectively cut the test chamber volume in half when the holes were stopped with silicone stoppers. The greatest depth of the test solution was about 13 cm, which yielded a maximum volume of approximately 10 L when one side was in use and approximately 20 L when both sides of the test chamber were in use.
- Retention baskets: Mysids were maintained within retention baskets to facilitate daily observations and enumeration. As retention baskets were not completely isolated from one another, these retention baskets were not replicates. Only the test chamber containing the retention baskets was considered a true replicate. Two cylindrical retention baskets, one containing mysids for the reproduction observations and the other for growth observations, were used in each replicate test chamber at the initiation of the F0-mysid exposure. Each growth and reproduction retention basket consisted of a glass Petri dish base (approximately 1.5 × 15 cm) with a nylon screen collar (Nitex mesh with an approximate mesh opening of 355 μm). Adult mysids from the reproduction retention baskets were paired at the time of sexual maturation and placed into brood cups consisting of a glass Petri dish base (approximately 1.5 × 10 cm) with the same type of nylon screen collar. The nylon screen for each retention basket and brood cup was attached to the Petri dish with translucent silicone sealant. Although the retention baskets and brood cups were not individually covered, the collars for each were tall enough to prevent the loss of mysids.
- Aeration: no
- Type of flow-through: proportional diluter
- Renewal rate of test solution: During the course of the definitive test, approximately 55 L of dilution-water control and test solution were delivered to each chamber each day while only one side of the test chamber was in use. This rate was sufficient to provide approximately 5.5 volume additions in a 24-hour period. When both sides were in use approximately 109 L of dilution-water control and test solution were delivered to each chamber each day. This rate was also sufficient to provide approximately 5.5 volume additions in a 24-hour period.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was a laboratory saltwater prepared by adding a commercial sea salt mix to laboratory freshwater at a target salinity 20 ± 2 ‰. The laboratory freshwater consists of wellwater 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 passed through a particulate filter and an ultraviolet steriliser.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Fluorescent lighting was set with a photoperiod of 14 hours of light and 10 hours of dark, with two 30-minute transition periods of lower light intensity to simulate dawn and dusk.
- Light intensity: The light measurements in the three chambers were 430, 569 and 666 lux.

EFFECT PARAMETERS MEASURED
Observations of mortality and sublethal responses F0-mysid generation were made daily for the duration of the testing period. Dead mysids were counted, differentiated by gender (if mature), and removed daily. Any missing mysids at the time of observation were noted as not found and were considered dead if not found on following observations days; that mortality was subsequently reflected back to the time that the mysid was first observed missing. Brood pouches were first evident on day 10 of the exposure in the control and all test material treatments. Any mysids that were inadvertently damaged or died as a result of becoming impinged onto the retention- or brood-basket mesh were removed from the initial number of mysids for those replicates and were not considered to be treatment-related mortality.

ENVIRONMENTAL PARAMETERS
Temperature, dissolved oxygen, and pH were measured in the replicate test chambers of all treatments at test initiation and termination and at least weekly during the definitive test. Temperature and pH were measured with a WTW Model 330i pH meter or WTW Multi 3500i multimeter. Dissolved oxygen was measured with a WTW OXi 330i dissolved oxygen meter or WTW Multi 3500i multimeter. Test solution salinity was measured daily in at least one test chamber. The salinity was measured with a WTW Model 330i conductivity/salinity meter. Light intensity at the level of the test solution was measured in three test chambers that ranged across the water bath with an LI-COR Model LI-189 light meter equipped with a photometric sensor on study day 28. Temperature was also recorded continuously in a centrally located test chamber by using an electronic data-logging system.

RANGE-FINDING STUDY
A flow-through range-finding test was conducted at nominal concentrations of 0 (control), 6.5, 13, 25, 50 and 100 mg a.i./L.
A total of 15 juvenile mysids (<24 hours old) were added to a retention chamber with one retention chamber per test chamber yielding 15 juvenile mysids per treatment group. The mysids were fed brine shrimp nauplii at least three times daily during the range-finding test, except at test termination when the mysids were fed twice.
After 19 days of exposure, the treatment mean percent survival of the mysids was 93, 93, 100, 87, 100 and 100 % in the 0 (control), 6.5, 13, 25, 50 and 100 mg a.i./L treatments, respectively. The highest number of females with brood pouches present was 6, 8, 8, 9, 5 and 7 in the 0 (control), 6.5, 13, 25, 50 and 100 mg a.i./L treatments, respectively. The number of young per female was 5.0, 7.9, 4.5, 4.8, 8.6 and 4.1 in the 0 (control), 6.5, 13, 25, 50 and 100 mg a.i./L treatments, respectively. The mean length of male mysids on day 19 was 5.58, 5.63, 5.52, 5.81, 5.69 and 5.61 mm in the control, 6.5, 13, 25, 50 and 100 mg a.i./L treatments, respectively. The mean length of female mysids on day 19 was 5.53, 5.68, 5.48, 5.58, 5.89 and 5.58 mm in the 0 (control), 6.5, 13, 25, 50 and 100 mg a.i./L treatments, respectively. Analytical confirmation of test solution samples collected on day 16 of the range-finding test from the 6.5, 25, and 100 mg a.i./L test material solutions determined these solutions ranged from 98-107 % of their nominal concentration.
The range-finding test data indicated no biologically significant adverse effects on survival, reproduction or growth in the highest nominal concentration tested (i.e., 100 mg a.i./L). Based on these range-finding test results the concentrations selected for the definitive test were 0 (control), 6.5, 13, 25, 50, and 100 mg a.i./L.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: survival, growth, and reproduction

Remarks on result:

other: There were no statistically significant differences between the control and test material treatments for all endpoints.

Details on results:

SURVIVAL
Mean survival of F0 mysids after 7 days of exposure was 100 % in the control and all test material treatments. Mean survival of F0 mysids after 13 days of exposure was 100 % in the control and ranged from 98 % in the 24.3 mg a.i./L treatment to 100 % in the 6.59, 13.8, 50.9, and 101 mg a.i./L treatments. After 14 days of exposure, mean survival of F0 mysids was 98 % in the control and ranged from 98 % in the 50.9 and 101 mg a.i./L treatments to 100 % in the 6.59, 13.8, and 24.3 mg a.i./L treatments. After 21 days of exposure, mean survival of F0 mysids was 98 % in the control and ranged from 91 % in the 50.9 mg a.i./L treatment to 100 % in the 6.59, 13.8, and 24.3 mg a.i./L treatments. After 28 days of exposure, mean survival of F0 mysids was 95 % in the control and ranged from 88 % in the 50.9 mg a.i./L treatment to 100 % in the 13.8 and 24.3 mg a.i./L treatments.

BODY LENGTH
The 28-day mean body length for the F0-male mysids was 6.12 mm in the control and ranged from 6.02 mm in the 50.9 mg a.i./L test material treatment to 6.13 mm in the 6.59 mg a.i./L test material treatment. The 28-day mean body length for the F0-female mysids was 6.29 mm in the control and ranged from 6.23 mm in the 13.8 and 101 mg a.i./L test material treatmentsto 6.41 mm in the 24.3 mg a.i./L test substance treatment. There was no statistically significant reduction in F0-male or female 28-day mean body lengths as compared to the control in any of the test-material treatments.

FIRST BROOD RELEASE
Brood pouches were first evident on day 10 of the exposure in the control and all test-material treatments. The mean day of first-brood release by F0 mysids was 16.5 days in the control and ranged from 16.3 days in the 6.59 mg a.i./L test material treatment to 16.8 days in the 24.3 and 101 mg a.i./L tests material treatments. There was no statistically significant delay in mean day of first-brood release between the control and test-material treatments.

YOUNG PRODUCED
The mean number of total young produced per female during the 28-day exposure was 31.6 young in the control and ranged from 28.4 young in the 101 mg a.i./L test material treatment to 35.3 young in the 24.3 mg a.i./L test material treatment. There was no statistically significant reduction in the number of young produced per female as compared to the control in any of the test-material treatments.

F1 SURVIVAL
Mean survival of F1 mysids after both 4 and 7 days of exposure was 100 % in the control and ranged from 98 % in the 13.8 and 24.3 mg a.i./L treatments to 100 % in the 6.59, 50.9, and 101 mg a.i./L treatments. After 11 days of exposure, mean survival of F1 mysids was 98 % in the control and ranged from 96 % in the 13.8 mg a.i./L treatment to 100 % in the 6.59 and 101 mg a.i./L treatments. There were no statistically significant reduction in F1-mysid survival rates on day 4, 7, and 11 in any of the test-material treatments as compared to the control.

F1 BODY LENGTH
The 11-day mean body length for the F1-male mysids was 4.64 mm in the control and ranged from 4.70 mm in the 24.3 mg a.i./L test-material treatment to 4.76 mm in the 50.9 mg a.i./L test-material treatment. The 11-day mean body length for the F1-female mysids was 4.78 mm in the control and ranged from 4.74 mm in the 6.59 mg a.i./L treatment to 4.89 mm in the 13.9 and 24.3 mg a.i/L treatments. There was no statistically significant reduction in F1-male or female mysid mean body length, as compared to the control in any of the test-material treatments.

Reported statistics and error estimates:

All statistical analyses were performed using SAS software (SAS version 9.1 for Windows). The NOECs, based on percent survival, survival of second generation offspring, reproduction (i.e., young per female) and adult length were estimated using a one-way analysis of variance (ANOVA) procedure and either one-tailed Fisher’s test with Hochberg’s familywise adjustment for significance or a one-tailed Dunnett's test. The alternate hypothesis was that the mean for the parameter in the treated exposures was reduced in comparison to the negative control mean for that same parameter. The time to first brood release was analysed using a one-tailed Dunnett’s test to determine significant enhancing effects on this parameter. For all analyses, prior to the Dunnett's test, a Shapiro-Wilk test for normality and Levene’s test for homogeneity of variance over treatments were conducted at each time point. When the p values from the Shapiro-Wilk’s and Levene’s test were greater than 0.01, indicating normality and insignificant heterogeneity, the analysis was performed on the raw value. A log transformation, or rank if these transformations were not adequate, was used when the p value was less than 0.01. The assumptions of normality and homogeneity of variance were met for all growth (i.e., body length) data; therefore, a parametric analysis was performed on these data. The assumptions of normality and homogeneity of variance were not met for all other parameters; therefore, a non-parametric analysis was performed on the ranks of the data. The maximum acceptable toxicant concentration (MATC), calculated as the square root of the product of the NOEC and LOEC concentrations for the most sensitive toxicological endpoint, could not be determined because the highest concentration tested was determined to be the NOEC. Median lethal concentration (LC50) values and their 95 % confidence limits could not be established because mortality was <50 % in all treatments.

Analytical Chemistry

The mean measured test concentrations of test material in the test-material treatments for the 28-day exposure were 6.59, 13.8, 24.3, 50.9, and 101 mg a.i./L and ranged from 97 % to 106 % of the nominal concentrations and ranged from 86 to 91 % of the day-0 measured concentrations. All but the day-21 measured concentrations were ± 20 % of the day-0 measured concentrations. The day-21 measured concentrations ranged from 73 to 75 % of the day-0 measured concentrations. No residues of test material above 0.297 mg a.i./L, the MQL (Method Quantification Limit) established during the definitive test, were detected in the control.

Water Quality Parameters

All measured water-quality parameters during the 28-day exposure were within the limits specified by the study protocol. Water temperature ranged from 24.2 to 25.0 °C. The dissolved oxygen values ranged from 6.46 to 7.70 mg/L or 90 to 97 % saturation. Since the dissolved oxygen levels remained above 60 % saturation, it was not necessary to use aeration to maintain the dissolved oxygen concentrations. The pH ranged from 7.72 to 8.08. Salinity ranged from 19.2 to 20.5 ‰

Validity criteria fulfilled:

yes

Conclusions:

There were no statistically significant differences between the control and test material treatments for all endpoints. Based on mean measured concentrations of test material during the 28-day exposure, the NOEC and LOEC values for day-28 F0-mysid survival, F0-male and F0-female mysid length on days 14 and 28, F0-mysid day of first brood, F0-mysid mean number of total young produced per female, F1-mysid survival on day 11, and F1-male and -female mysid length on days 4, 7, and 11, were 101 and >101 mg a.i./L.

Executive summary:

The chronic toxicity of the test material was investigated in a life-cycle toxicity test with the saltwater Mysid, Americamysis bahia. The study was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPPTS 850.1350 and EPA OPP 72-4.

During the study the mysids were exposed to test concentrations of test material of 0 (control), 6.5, 13, 25, 50, and 100 mg a.i./L, under flow-through conditions. Diluter stock solutions were prepared at a target concentration of 983 mg a.i./L in dilution water. An LMI pump introduced 400-mL volumes of the diluter stock solution to the diluter system, where the diluter stock solution volume was diluted with approximately 3,500 mL of dilution water.

Mysids were fed ad libitum brine shrimp nauplii (Artemia sp.; 24 - 48 hours old) at least two times daily. Observations of mortality and sublethal responses F0-mysid generation were made daily for the duration of the testing period. The number of females with brood pouches was enumerated from the time brood pouches were first noted (day 10) until adults were paired on day 13. The body lengths of mysids (as measured by total midline body length) were measured to the nearest 0.1 mm with a dissecting microscope.

Ovigerous F0-female mysids (i.e., females with eggs within the marsupium) were isolated and paired with adult males and transferred to the brood baskets on day 13 of the exposure. Once paired, the mysids in the brood cups were observed for mortality and reproduction (i.e., young per female). The first day young were observed was considered the day of first brood, although release of these young may have occurred over 2 or 3 days. After 14 days of exposure, the body length of all surviving F0 mysids present in the growth-retention basket was measured. The growth-retention baskets were terminated following these measurements. The F1-mysid exposure phase of the test was initiated with the first 15 post-larval F1 mysids, or fewer when 15 young were not available. The post-larval F1 mysids were assigned to retention baskets within the same test chambers as the F0-mysid exposure. The isolated F1 mysids were observed daily for mortality during the exposure and when F1 mysids were 11 days old. The F1 mysids were terminated when they reached 11 days of age because this was the maximum achievable age for all F1 mysids at termination of the F0-mysid exposure (i.e., study day 28). The body length of all surviving 11-day old F1 mysids was measured.

Temperature, dissolved oxygen, salinity, and pH were measured in the replicate test chambers of all treatments at test initiation and termination and at least weekly during the definitive test. Test solution salinity was also measured daily in at least one replicate test chamber. Light intensity was measured on day 28.

There were no statistically significant differences between the control and test material treatments for all endpoints. Based on mean measured concentrations of test material during the 28-day exposure, the NOEC and LOEC values for day-28 F0-mysid survival, F0-male and F0-female mysid length on days 14 and 28, F0-mysid day of first brood, F0-mysid mean number of total young produced per female, F1-mysid survival on day 11, and F1-male and -female mysid length on days 4, 7, and 11, were 101 and >101 mg a.i./L.

Description of key information

FRESHWATER
NOEC (survival, reproduction and growth) = 100 mg/L, Daphnia magna Straus, OECD 211, EPA OPP 72-4, Henry et al. (2003)
SALTWATER
NOEC (survival, growth, reproduction) = 100 mg/L, Americamysis bahia, EPA OPPTS 850.1350, EPA OPP 72-4, Hicks (2011)

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:

100 mg/L

Additional information

Two studies investigating the long-term toxicity of the substance to aquatic invertebrates 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 Henry et al. (2003) the chronic toxicity of the test material was investigated in a life-cycle toxicity test with the saltwater Mysid, Americamysis bahia. The study was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPPTS 850.1350 and EPA OPP 72-4.

During the study the mysids were exposed to test concentrations of test material of 0 (control), 6.5, 13, 25, 50, and 100 mg a.i./L, under flow-through conditions. Diluter stock solutions were prepared at a target concentration of 983 mg a.i./L in dilution water. An LMI pump introduced 400-mL volumes of the diluter stock solution to the diluter system, where the diluter stock solution volume was diluted with approximately 3,500 mL of dilution water.

Mysids were fed ad libitum brine shrimp nauplii (Artemia sp.; 24 - 48 hours old) at least two times daily. Observations of mortality and sublethal responses F0-mysid generation were made daily for the duration of the testing period. The number of females with brood pouches was enumerated from the time brood pouches were first noted (day 10) until adults were paired on day 13. The body lengths of mysids (as measured by total midline body length) were measured to the nearest 0.1 mm with a dissecting microscope.

Ovigerous F0-female mysids (i.e., females with eggs within the marsupium) were isolated and paired with adult males and transferred to the brood baskets on day 13 of the exposure. Once paired, the mysids in the brood cups were observed for mortality and reproduction (i.e., young per female). The first day young were observed was considered the day of first brood, although release of these young may have occurred over 2 or 3 days. After 14 days of exposure, the body length of all surviving F0 mysids present in the growth-retention basket was measured. The growth-retention baskets were terminated following these measurements. The F1-mysid exposure phase of the test was initiated with the first 15 post-larval F1 mysids, or fewer when 15 young were not available. The post-larval F1 mysids were assigned to retention baskets within the same test chambers as the F0-mysid exposure. The isolated F1 mysids were observed daily for mortality during the exposure and when F1 mysids were 11 days old. The F1 mysids were terminated when they reached 11 days of age because this was the maximum achievable age for all F1 mysids at termination of the F0-mysid exposure (i.e., study day 28). The body length of all surviving 11-day old F1 mysids was measured.

Temperature, dissolved oxygen, salinity, and pH were measured in the replicate test chambers of all treatments at test initiation and termination and at least weekly during the definitive test. Test solution salinity was also measured daily in at least one replicate test chamber. Light intensity was measured on day 28.

There were no statistically significant differences between the control and test material treatments for all endpoints. Based on mean measured concentrations of test material during the 28-day exposure, the NOEC and LOEC values for day-28 F0-mysid survival, F0-male and F0-female mysid length on days 14 and 28, F0-mysid day of first brood, F0-mysid mean number of total young produced per female, F1-mysid survival on day 11, and F1-male and -female mysid length on days 4, 7, and 11, were 101 and >101 mg a.i./L.