Diquat dibromide

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:

20 Jul 2012 to 30 Aug 2012

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)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Version / remarks:

1996 (draft)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Water samples were collected from one test chamber of each treatment and control group two days prior to test initiation to confirm the operation of the diluter. Water samples were collected from alternating replicate test chambers of each treatment and control group on Days 0, 7, 14, 20, 28 and 34 (test termination) to determine concentrations of the test substance in the test chambers. On Day 21 of the test, additional samples of test solutions were collected and analyzed to confirm the test solutions concentrations when the results from Day 20 were suspect. In addition, on Day 21, samples were also collected from all replicates of each treatment and control groups and stored as backup samples for possible future analysis. On Day 22, additional samples were collected and analyzed to confirm the test concentrations in the test chambers after a power interruption caused a brief interruption in the delivery of stock solutions. All samples were collected at mid-depth in the test chambers, placed in glass vials and processed immediately for analysis.

Vehicle:

no

Details on test solutions:

Stock solutions were prepared three times during the study. A primary test substance stock solution was prepared in reverse osmosis (RO) water at a nominal concentration of 100 mg/mL test substance cation (187 mg/L pure test substance). Proportional dilutions of the primary stock were made in RO water to prepare additional stock solutions at nominal concentrations of 2.570, 6.433, 15.73 and 40.00 mg/mL cation (4.8, 12.0, 29.4 and 74.7 mg/L pure test substance). The stock solutions were mixed by inversion and ranged in appearance from light beige in color to dark amber, with color intensity increasing with increasing concentration. No evidence of precipitate was noted in any of the stock solutions. Stock solutions were stored under refrigerated conditions and fresh aliquots were placed in the syringe pumps every one or two days during the test. The stock solutions were delivered
to the diluter mixing chambers (at a rate of 14 µL/minute) where they were mixed with dilution water (at a rate of 200 mL/minute) to achieve the desired test concentrations of 0.18, 0.45, 1.1, 2.8 and 7.0 mg/L cation (0.34, 0.84, 2.1, 5.2 and 13 mg/L pure test substance). The resultant test concentrations were adjusted for the purity of the cation in the test substance (21.5% w/w). The test substance equivalents were calculated based on the ratio of the molecular weights of the test substance (344.0) and its cation (184.2).

Test organisms (species):

Cyprinodon variegatus

Details on test organisms:

TEST ORGANISM
- Common name: Sheepshead minnow
- Health of the organisms source: The embryos arrived free floating and were examined under a dissecting microscope to select healthy viable specimens at approximately the same stage of development.
- Age at study initiation: Approximately 25 hours post-fertilization
- Feeding during test : Newly-hatched larvae were fed live brine shrimp nauplii (Artemia sp.) three times per day during the test. Fish were not fed for at least 48 hours prior to the termination of the test to allow for clearance of the digestive tracts before weight measurements were made. To ensure that the feeding rate per fish remained constant, rations were adjusted at least weekly to account for losses due to mortality. Excess feed was siphoned from the test chambers periodically during the test.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

34 d

Remarks on exposure duration:

6 days Hatch and 28 days Post-Hatch

Test temperature:

25 ± 1 °C

pH:

7.7 - 7.9

Dissolved oxygen:

6.8 - 7.4 mg O2/L

Salinity:

20 - 21‰

Nominal and measured concentrations:

- Nominal concentration: 0 (negative control), 0.34, 0.84, 2.1, 5.2 and 13 mg/L
- Measured concentration: < LOQ (negative control), 0.34, 0.80, 2.2, 5.6 and 14 mg/L, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: 9 L glass aquaria
- Filled volume of the test vessel: Approximately 7 L of test solution (The depth of the test water in a representative test chamber was 15.6 cm. )
- Type of flow-through: Continuous-flow diluter.
Syringe pumps were used to deliver the five test substance stocks into mixing chambers assigned to each treatment. The syringe pumps were calibrated prior to the test. The stock solutions were diluted with saltwater in the mixing chambers to obtain the desired test concentrations. The flow of dilution water to the mixing chambers was controlled by rotameters, which were calibrated prior to test initiation and verified at approximately weekly intervals during the test. The flow of test water from each mixing chamber was split and allowed to flow into four replicate test chambers. The proportion of the test water that was split into each replicate was checked prior to the test and approximately weekly during the test to ensure that flow rates varied by no more than ±10% of the mean for the four replicates. The diluter flow rate was adjusted to provide approximately ten volume additions of test water to each test chamber per day. The general operation of the diluter was checked visually at least two times per day during the test and at least once at the beginning and end of the test. Periodically during the test, all organisms were transferred to clean test chambers to prevent the buildup of bacterial/fungal growth
- No. of organisms per vessel: 20
- No. of vessels per concentration: 1
- No. of vessels per control: 1
Embryos were held in incubation cups constructed from glass cylinders 50 mm in diameter with 425 µm nylon screen attached to the bottom with silicone sealant. One cup was suspended in the water column of each test chamber and attached to a rocker arm. The reciprocating motion of the rocker arm (2 rpm) facilitated circulation of test water around the embryos during incubation.
- Biomass loading rate: Biomass loading at the end of the test, based on the negative control mean wet weight, was 0.02 g of fish per liter of test solution that passed through the test chamber during a 24-hour period. Instantaneous loading (the total wet weight of fish per liter of water in the tank) at the end of the test was 0.21 g fish/L.

TEST MEDIUM
- Source/preparation of dilution water: The water used for testing was natural seawater collected at Indian River Inlet, Delaware. The freshly-collected seawater was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800 L storage tank. The filtered saltwater then was diluted to a salinity of approximately 20‰ with freshwater from a well on the test facility site and was aerated with spray nozzles. Prior to use, the 20‰ water was filtered to 0.45 µm to remove fine particles and was passed through an ultraviolet (UV) sterilizer. Means and ranges of salinity and pH measurements taken during the four week period immediately preceding the test.

WATER PARAMETERS
- Temperature: Temperature was measured in each test chamber at the beginning of the test, weekly during the test, and at the end of the test using a liquid-in-glass thermometer. Temperature also was monitored continuously in one negative control replicate.
- Dissolved oxygen and pH: Dissolved oxygen and pH were measured in alternating replicates of each treatment and control group at the beginning of the test, weekly during the test, and at the end of the test.
- Salinity: Salinity was measured in one alternating replicate of the negative control and the highest concentration at the beginning of the test, weekly during the test and at test end.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting.
- Light intensity:

EFFECT PARAMETERS MEASURED
During the first day of exposure, embryos were observed twice for mortality and for eggs with fungus. Thereafter, until hatching was complete, observations of embryo mortality and the removal of dead embryos were performed once daily. When hatching reached > 90% in the control groups on Day 6 of the test, the larvae were released to their respective test chambers and the post-hatch period began. Any unhatched embryos were kept in the egg cups until they hatched and were released into the test chamber, or until death of the embryo occurred. During
the 28-day post-hatch exposure period, the larvae were observed daily to evaluate the number of mortalities and the number of individuals exhibiting clinical signs of toxicity or abnormal behavior. From these observations, time to hatch, hatching success, and post-hatch growth and survival were evaluated. Hatching success was calculated as the percentage of embryos that hatched successfully. Post-hatch survival was calculated as the number of larvae surviving to test termination divided by the total number of embryos, which hatched successfully. Post-hatch growth of the sheepshead minnows was evaluated at the conclusion of the 28-day post-hatch exposure period. Total length for each surviving fish was measured to the nearest 1 mm using a metric ruler, and wet and dry weights were measured to the nearest 0.1 mg using
an analytical balance. Fish were placed in an oven at approximately 60 °C for approximately 53 hours to obtain dry weight data.

Reference substance (positive control):

no

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

5.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: pure test substance

Basis for effect:

other: Growth (total length and wet weight)

Duration:

34 d

Dose descriptor:

LOEC

Effect conc.:

14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: pure test substance

Basis for effect:

other: Growth (total length and wet weight)

Details on results:

An overview of the results is provided in Table 1 in 'Any other results incl. tables'.

- Time to Hatch and Hatching Success: All viable embryos in the control, 0.34, 0.77, 2.2, 5.8 and 14 mg test substance/L replicates hatched by Days 6 or 7 of the test, and larvae were released into the test chambers on Day 6 of the test when > 90% of the viable embryos in the negative control replicates hatched. Daily observations of all the embryos indicated that there were no apparent differences in time to hatch between the control group and any of the test substance treatment groups. Hatching success in the negative control group was 98%. Hatching success in the 0.34, 0.77, 2.2, 5.8 and 14 mg/L treatment groups was 98, 99, 100, 100 and 96%, respectively. Fisher’s Exact test showed that there were no statistically significant decreases in hatching success at any of the test substance treatment groups when compared to the negative control (p > 0.05). Therefore, the NOEC for hatching success was 14 mg test substance/L and the LOEC was greater than 14 mg test substance/L.

- Larval Survival and Clinical Observations: Larval survival in the negative control group at test termination was 94%. Larval survival in the 0.34, 0.77, 2.2, 5.8 and 14 mg test substance/L treatment groups was 94, 94, 94, 98 and 94%, respectively. Fisher’s Exact test showed that there were no statistically significant decreases in larval survival noted in any of the test substance treatment groups when compared to the negative control data (p > 0.05). Consequently, the NOEC and LOEC based on percent larval survival at test termination were 14 and greater than 14 mg /L, respectively. The majority of the fish in the negative control and the 0.34, 0.77, 2.2, 5.8 and 14 mg /L treatment groups appeared normal throughout the test, with occasional observations of small fish. Occasional observations of small, lethargic, lying on the bottom of the test chamber and/or morphologically deformity (curled) were made in the negative control as well as in the 0.34 to 5.6 mg/L treatment groups. The observations of small and/or lethargic fish in the cation treatment groups were generally restricted to a few individuals and were generally comparable to the negative control group. Therefore, none of the sublethal effects noted for the fish in the 0.34 to 5.6 mg/L treatment groups were considered to be treatment-related. However, the number of small fish noted in the 14 mg/L treatment group was higher than the negative control and the rest of the treatment concentrations.

- Growth: Statistically significant reductions in mean total length and mean wet weight were noted among fish in the 14 mg/L treatment group in comparison to the negative control (Dunnett’s one-tailed test, p ≤ 0.05). No statistically significant reductions in mean dry weight were noted in any of the test substance treatment groups in comparison to the negative control (Dunnett’s one tailed test, p > 0.05). Consequently, the NOEC was 5.6 mg test substance/L and the LOEC was 14 mg test substance/L, based on growth.

Reported statistics and error estimates:

See statistical analysis in 'Any other information on materials and methods incl. tables'.

Table 1. Summary of Hatching Success, Larval Survival and Growth of Sheepshead Minnows Exposed to the test substance.

Mean Measured Test Concentration (mg test substance/L)	Percent Hatching Success^1	Percent Survival to Day 28 Post-Hatch^1	Day 28 Post-Hatch
			Mean Total Length ± SD (mm)	Mean Wet Weight ± SD (mg)	Mean Dry Weight ± SD (mg)^1
Negative Control	98	94	18.5 ± 0.24	72.7 ± 1.6	16.0 ± 0.15
0.34	98	94	18.2 ± 0.34	69.8 ± 3.9	15.0 ± 0.76
0.80	99	94	18.2 ± 0.17	70.0 ± 3.8	15.3 ± 0.81
2.2	100	94	18.5 ± 0.14	72.5 ± 2.0	16.0 ± 0.68
5.6	100	98	18.6 ± 0.19	74.0 ± 3.6	16.1 ± 0.63
14	96	94	17.3 ± 0.22*	65.3 ± 1.4*	15.1 ± 0.34

*          Statistically significant differences in growth (measured as total length and wet weight) using Dunnett’s one-tailed test, p ≤ 0.05.

1         There were no statistically significant reductions in hatching success, survival or growth (measured as dry weight) noted in any of the test substance treatment groups in comparison to the negative control (using Fisher’s Exact test, p > 0.05 for hatching success and survival and using Dunnett’s one-tailed test, p > 0.05 for growth).           

Validity criteria fulfilled:

yes

Remarks:

See Conditions for the Validity of the Test in 'Any other information on materinals and methods incl. tables'.

Conclusions:

Based on the findings, the 34-day NOEC for growth (total length and wet weight) was determined to be 5.6 mg test substance/L.

Executive summary:

To determine the chronic toxicity of the test substance in the early life-stage of fish, Sheepshead minnows (Cyprinodon variegatus) embryos (approximately 25 hours post-fertilization) were exposed to a geometric series of five test concentrations and a negative control (dilution water) under flow-through conditions.The exposure period included a 6-day embryo hatching period and a 28-day post-hatch juvenile growth period. This study was conducted in accordance with OECD TG 210 and EPA guideline OPPTS 850.1400. The study was in compliance with GLP criteria.

Five mean measured test concentrations of the test substance were used including 0.34, 0.77, 2.2, 5.8 and 14 mg/L (measured by HPLC). The study was carried out at 25 ± 1 °C, pH 7.7 - 7.9, and salinity 20 - 21‰.The dissolved oxygen concentration during the test was 6.8 - 7.4 mg O2/L. Observations of the effects of the test substance on time to hatch, hatching success, growth, and survival were used to calculate the NOEC and LOEC.

All viable embryos in the control and exposure replicates hatched by Days 6 or 7 of the test, and larvae were released into the test chambers on Day 6 of the test when > 90% of the viable embryos in the negative control replicates hatched.Daily observations of all the embryos indicated that there were no apparent differences in time to hatch between the control group and any of the treatment groups. Hatching success in the negative control group was 98%. Hatching success in the treatment groups was 96 - 100%. Fisher’s Exact test showed that there were no statistically significant decreases in hatching success at any of the test substance treatment groups when compared to the negative control (p > 0.05). 

Larval survival in the negative control group at test termination was 94% and 94 to 98%in the treatment groups. Fisher’s Exact test showed that there were no statistically significant decreases in larval survival noted in any of the test substance treatment groups when compared to the negative control data (p > 0.05). The majority of the fish in the negative control and treatment groups appeared normal throughout the test, with occasional observations of small fish. Occasional observations of small, lethargic, lying on the bottom of the test chamber and/or morphologically deformity (curled) were made in the negative control as well as in the 0.34 to 5.6 mg/L treatment groups. The observations of small and/or lethargic fish in the cation treatment groups were generally restricted to a few individuals and were generally comparable to the negative control group. Therefore, none of the sublethal effects noted for the fish in the 0.34 to 5.6 mg/L treatment groups were considered to be treatment-related. However, the number of small fish noted in the 14 mg /L treatment group was higher than the negative control and the rest of the treatment concentrations.

Statistically significant reductions in mean total length and mean wet weight were noted among fish in the 14 mg/L treatment group in comparison to the negative control (Dunnett’s one-tailed test, p ≤ 0.05). No statistically significant reductions in mean dry weight were noted in any of the test substance treatment groups in comparison to the negative control (Dunnett’s one tailed test, p > 0.05). Consequently, the NOEC was 5.6 mg test substance/L and the LOEC was 14 mg test substance/L, based on growth.

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Apr 1987 to 31 May 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The high and low test concentrations were sampled once before the definitive exposure began and analyzed for cation. Results of the analyses of these samples were used to judge whether sufficient quantities of the test material were being delivered and maintained in the exposure: aquaria to initiate the definitive embryo - larvae test. During the definitive test, samples were removed from one replicate of all test solutions and the control on test days 0, 3, 7, and weekly thereafter until, and including, test termination. Alternate replicate vessels were measured at each sampling interval. Each test solution sample was collected from the approximate midpoint of the aquarium with a volumetric pipet.

Vehicle:

no

Details on test solutions:

Diluter stock solutions were prepared every 5 - 6 days by adding 8.535 g of the test substance concentrate to 50 mL of distilled water. The resulting stock solution contained 170.7 mg/mL of the test substance.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Age at study initiation: < 24 hours after fertilization

FEEDING DURING TEST (Larvae)
- Food type: Live brine shrimp (Artemia salina) nauplii (The food used during this study was not analyzed for potential contaminants.)
- Frequency: Two times daily

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

34 d

Remarks on exposure duration:

4 days hatch and 30 days post-hatch

Hardness:

32 - 34 mg/L as CaCO3

Test temperature:

25 °C

pH:

6.7 - 7.4

Dissolved oxygen:

7.2 - 7.5 mg O2/L

Nominal and measured concentrations:

- Nominal concentration: 0 (negative control), 0.094, 0.19, 0.38, 0.75, 1.5 and 3.0 mg/L
- Measured concentration: < LOQ (negative control), 0.085, 0.13, 0.30, 0.58, 1.5 and 3.1 mg/L, respectively.

Details on test conditions:

TEST SYSTEM
Prior to hatching ( day 0 - day 4)
- Test vessel: Glass jars
- Size of vessel: 5 cm O.D., 8 cm high with 40-mesh screen bottoms.
- No. of organisms per vessel: 60
- No. of vessels per concentration: 2
- No. of vessels per control: 2

Post-hatching (day 4 - day 34)
- Test vessel: Glass aquarium
- Size of vessel: 39 x 20 x 25 cm with a 19.5 cm high side drain that maintained a constant test water volume of 15 L
- No. of organisms per vessel: 40
- No. of vessels per concentration: 2
- No. of vessels per control: 2

- Type of flow-through: Proportional diluter
- Flow rate: 0.5 L of test water to each aquarium at an average rate of 180 times per day (equivalent to approximately six aquarium volume
replacements per 24-hour period, with a 90% replacement time- of approximately 9 hours)
- Dilution factor: 0.50
A 50 mL gas tight syringe with a stainless steel needle, extended with polyethylene tubing, was mechanically activated during each diluter cycle to inject 0.0348 mL of the 170.7 mg/mL stock solution into the diluter's chemical mixing chamber containing 1.98 L of dilution water. The mixing chamber was positioned over a magnetic stirrer which continuously mixed the contents. The 1.98 L solution contained in the mixing chamber constituted the highest test concentration (nominal concentration of 3.0 mg/L test substance) and was subsequently proportionally diluted (50%) to provide the range of exposure concentrations.

TEST MEDIUM
- Source/preparation of dilution water: The dilution and control water was well water which was pumped into an epoxy-coated concrete reservoir where it was supplemented with Town of Wareham untreated well water and aerated. The characterization of the well water was checked weekly.
- Total hardness: 30 - 38 mg/L as CaCO3
- Alkalinity: 28 - 32 mg/L as CaCO3
- pH: 7.0 - 7.1
- Conductivity: 110 - 120 µmhos/cm

WATER PARAMETERS
Dissolved oxygen concentration, pH and temperature were measured in every aquarium on day 0. Subsequently, these parameters were measured daily in one replicate aquarium of each treatment level and the control such that each aquarium was measured on alternating days.Total hardness as CaCO3 was measured on day 0 and weekly thereafter in alternating replicates of the high and low test concentrations and the control.

OTHER TEST CONDITIONS
Aquaria were brushed and siphoned when necessary (generally several times per week) to remove excess food and fecal matter.
- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: 10 - 40 footcandles (at water surface)

EFFECT PARAMETERS MEASURED
Dead embryos were counted daily until hatching was complete. Hatching was deemed complete (exposure day 4) when no more than 5 unhatched viable embryos remained in any treatment level. Calculations of percentage survival of organisms at hatch were based on the number of live larvae and embryos per incubation cup after hatching was completed compared to the number of embryos per cup on test day 0.
Behavior and appearance of 'larvae were observed and recorded daily, and larval survival was estimated twice weekly. At 30 days post-hatch the larvae from each aquarium were anesthetized with approximately 500 mg/L of tricaine methanesulfonate, and percentage survival, mean total length, and mean wet weight were determined. The larvae were measured and weighed individually to calculate the mean and stadard deciation of total ength and wet weight.

RANGE-FINDING STUDY
Prior to initiating the definitive early life stage study, preliminary tests exposing fathead minnow embryos and larvae were conducted. During the preliminary tests all exposed fathead minnows died from exposure to the test substance concentrations ≥ 7.5 mg/L. The preliminary test exposing larvae for 14 days resulted in mortality of 40% of the organisms exposed to 3.7 mg/L and 0% mortality among organisms exposed to the test substance concentrations ≤ 1.9 mg/L. Based on these preliminary data nominal concentrations of 0.094, 0.19, 0.38, 0.75, 1.5 and 3.0 mg/L were selected for the definitive early life stage exposure.

Reference substance (positive control):

no

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

0.24 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: pure test substance

Basis for effect:

weight

Remarks:

wet weight

Remarks on result:

other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation.

Duration:

34 h

Dose descriptor:

NOEC

Effect conc.:

0.58 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

wet weight

Remarks on result:

other: Original value presented in study

Details on results:

An overview of the results is provided in Table 1 in 'Any other information on results incl. tables'.

- Survival: Exposure to all test substance concentrations tested (0.085 - 3.1 mg/L) did not adversely affect the survival of organisms at hatch (day 4) or larval survival at test termination. Mean survival of organisms at hatch ranged from 85 to 93% among organisms exposed to all treatment levels and the control with no apparent concentration - dependent effects. At test termination larval survival in the highest test concentration (3.1 mg/L) was slightly reduced (84%) but statistically comparable to
the survival of the control organisms (91%). The results of the preliminary tests established that exposure concentrations approximately 2X the highest concentrations tested during the definitive study (3.1 mg/L) significantly affected (reduction) the survival of juvenile fathead minnow. Based on these data the MATC for the test substance and fathead minnow larval survival can be estimated as approximately 4 - 5 mg/L (estimated geometric mean MATC).

- Larval growth: A reduction in larval growth (measured as wet weight) during the 30 - day post hatch period was the most sensitive indicator of the toxicity of the test substance to fathead minnow embryos and larvae. The mean wet weight of larvae in the two highest treatment levels (3.1 and 1.5 mg/L) were 250 and 304 mg, respectively, which was significantly less than the weight of the control larvae (342 mg). Larval length was significantly reduced in the highest test concentration (3.1 mg/L). The mean wet weight of larvae exposed to the test substance concentrations < 0.58 mg/L was statistically comparable to the control larvae growth, and 4 illustrate the observed reduction in larval growth in the two highest treatment levels of the test substance tested and their
relation to the other tested concentrations and the control.

Based on the significantly (P < 0.05) reduced growth (measured as wet weight) of larvae exposed to the test material concentrations of 3.1 and 1.5 mg/L, the NOEC for growth was determinded to be 0.58 mg/L. The maximum acceptable toxicant concentration ( MATC) of this test material for fathead minnow embryos and larvae is estimated to be ≥ 0.58 mg/L and ≤ 1.5 mg/L ( geometric mean MATC = 0.93 mg/L).

Reported statistics and error estimates:

See 'Statistical Analysis' in 'Any other information on materials and methods incl. tables'.

Table 1. Survival of organisms at hatch (test day 4) and survival, total length and wet weight of fathead minnow (Pimephales promelas) larvae after 30 days post-hatch exposure to the test substance.

Mean measured concentration (mg/L)		Survival of organisms at hatch (%)	Larvae (30 dyas post-hatch)
			Larvae survival (%)	Mean total lenth (SD) (mm)	Mean wet weight (SD) (mg)
3.1	A	90	83	30 (2.9)	247 (72)
	B	85	85	29 (4.3)	253 (89)
	Mean	88	84	29 (3.7)^a	250 (80)^a
1.5	A	93	98	31 (2.8)	285 (87)
	B	88	78	33 (2.3)	328 (76)
	Mean	91	88	32 (2.7)	304 (85)^a
0.58	A	87	95	32 (3.8)	310 (99)
	B	88	95	32 (2.2)	314 (93)
	Mean	88	95	32 (3.1)	312 (95)
0.3	A	92	95	33 (2.9)	345 (100)
	B	93	93	32 (4.0)	308 (100)
	Mean	93	94	32 (3.5)	327 (101)
0.13	A	97	93	33 (2.1)	325 (66)
	B	87	88	33 (3.5)	343 (92)
	Mean	92	90	33 (2.9)	334 (80)
0.085	A	85	95	32 (2.7)	304 (84)
	B	85	88	32 (2.5)	313 (73)
	Mean	85	91	32 (2.6)	308 (79)
Control	A	90	90	33 (2.0)	326 (75)
	B	85	93	34 (3.1)	358 (107)
	Mean	88	91	33 (2.6)	342 ( 93)

a. Indicates significantly different (P < 0.05) from the control data.

 

Calculation of key result

The original effect levels were expressed as aqueous solution (41.4% purity) of the monohydrate form of the registered substance. The key effect levels are re-calculated to reflect the anhydrous form of the test substance corrected for the amount of water.:

41.4% x 0.58 mg/L = 0.24 mg/L

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 34-day NOEC for growth (measured as wet weight) was determined to be 0.58 mg test material/L, equivalent to 0.24 mg pure test substance/L.

Executive summary:

To determine the chronic toxicity of the test substance to early life-stage of fish, Fathead minnow (Pimephales promelas) embryos and larvae were continuously exposed for 34 days (30 days post hatch) under a flow-through system. The study was conducted in accordance with EPA 72 -4 guideline and in compliance with GLP. The mean measured concentration of the test material were 0.085, 0.13, 0.30, 0.58, 1.5 and 3.1 mg/L (measured by a spectrophotometry; nominal concentrations were 0.094, 0.19, 0.38, 0.75, 1.5 and 3.0 mg/L, respectively). The study was carried out in 25°C, pH 6.7 – 7.4, dissolved oxygen concentration of 7.2 - 7.5 mg O2/L and water hardness of 32 - 34 mg/L (as CaCO3) condition. Observations were made on survival of organisms at hatch and survival and growth (wet weight and total length) of larvae after 30 days post hatch exposure. Exposure to all test substance concentrations tested (0.085 - 3.1 mg/L) did not adversely affect the survival of organisms at hatch (day 4) or larval survival at test termination. Mean survival of organisms at hatch ranged from 85 to 93% among organisms exposed to all treatment levels and the control with no apparent concentration - dependent effects. At test termination larval survival in the highest test concentration (3.1 mg/L) was slightly reduced (84%) but statistically comparable to the survival of the control organisms (91%). The results of the preliminary tests established that exposure concentrations approximately 2X the highest concentrations tested during the definitive study (3.1 mg/L) significantly affected (reduction) the survival of juvenile fathead minnow. Based on these data the MATC for the test substance and fathead minnow larval survival can be estimated as approximately 4 - 5 mg/L (estimated geometric mean MATC). A reduction in larval growth (measured as wet weight) during the 30 - day post hatch period was the most sensitive indicator of the toxicity of the test substance to fathead minnow embryos and larvae. The mean wet weight of larvae in the two highest treatment levels (3.1 and 1.5 mg/L) were 250 and 304 mg, respectively, which was significantly less than the weight of the control larvae (342 mg). Larval length was significantly reduced in the highest test concentration (3.1 mg/L). The mean wet weight of larvae exposed to the test material concentrations < 0.58 mg/L was statistically comparable to the control larvae growth, and 4 illustrate the observed reduction in larval growth in the two highest treatment levels of the test substance tested and their relation to the other tested concentrations and the control. Based on the findings, the 34-day NOEC for growth is determined to be 0.58 mg test substance/L, equivalent to 0.24 mg test substance/L.

Description of key information

Freshwater, 34-d NOEC = 0.24 mg test substance/L, growth (wet weight), Pimephales promelas, EPA 72- 4, Surprenant 1987

Marine water, 34-d NOEC = 5.6 mg test substance/L, growth (total length and wet weight), Cyprinodon variegatus, OECD TG 210, Minderhout 2012

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

0.24 mg/L

Marine water fish

Marine water fish

Dose descriptor:

NOEC

Effect concentration:

5.6 mg/L

Additional information

Freshwater

To determine the chronic toxicity of the test substance to early life-stage of fish, Fathead minnow (Pimephales promelas) embryos and larvae were continuously exposed for 34 days (30 days post hatch) under a flow-through system. The study was conducted in accordance with EPA 72 -4 guideline and in compliance with GLP. The mean measured concentration of the test material were 0.085, 0.13, 0.30, 0.58, 1.5 and 3.1 mg/L (measured by a spectrophotometry; nominal concentrations were 0.094, 0.19, 0.38, 0.75, 1.5 and 3.0 mg/L, respectively). The study was carried out in 25°C, pH 6.7 – 7.4, dissolved oxygen concentration of 7.2 - 7.5 mg O2/L and water hardness of 32 - 34 mg/L (as CaCO3) condition. Observations were made on survival of organisms at hatch and survival and growth (wet weight and total length) of larvae after 30 days post hatch exposure. Exposure to all test substance concentrations tested (0.085 - 3.1 mg/L) did not adversely affect the survival of organisms at hatch (day 4) or larval survival at test termination. Mean survival of organisms at hatch ranged from 85 to 93% among organisms exposed to all treatment levels and the control with no apparent concentration - dependent effects. At test termination larval survival in the highest test concentration (3.1 mg/L) was slightly reduced (84%) but statistically comparable to the survival of the control organisms (91%). The results of the preliminary tests established that exposure concentrations approximately 2X the highest concentrations tested during the definitive study (3.1 mg/L) significantly affected (reduction) the survival of juvenile fathead minnow. Based on these data the MATC for the test substance and fathead minnow larval survival can be estimated as approximately 4 - 5 mg/L (estimated geometric mean MATC). A reduction in larval growth (measured as wet weight) during the 30 - day post hatch period was the most sensitive indicator of the toxicity of the test substance to fathead minnow embryos and larvae. The mean wet weight of larvae in the two highest treatment levels (3.1 and 1.5 mg/L) were 250 and 304 mg, respectively, which was significantly less than the weight of the control larvae (342 mg). Larval length was significantly reduced in the highest test concentration (3.1 mg/L). The mean wet weight of larvae exposed to the test material concentrations < 0.58 mg/L was statistically comparable to the control larvae growth, and 4 illustrate the observed reduction in larval growth in the two highest treatment levels of the test substance tested and their relation to the other tested concentrations and the control. Based on the findings, the 34-day NOEC for growth is determined to be 0.58 mg test substance/L, equivalent to 0.24 mg test substance/L.

 

Marine water

To determine the chronic toxicity of the test substance in the early life-stage of fish, Sheepshead minnows (Cyprinodon variegatus) embryos (approximately 25 hours post-fertilization) were exposed to a geometric series of five test concentrations and a negative control (dilution water) under flow-through conditions.The exposure period included a 6-day embryo hatching period and a 28-day post-hatch juvenile growth period. This study was conducted in accordance with OECD TG 210 and EPA guideline OPPTS 850.1400. The study was in compliance with GLP criteria.

Five mean measured test concentrations of the test substance were used including 0.34, 0.77, 2.2, 5.8 and 14 mg/L (measured by HPLC). The study was carried out at 25 ± 1 °C, pH 7.7 - 7.9, and salinity 20 - 21‰.The dissolved oxygen concentration during the test was 6.8 - 7.4 mg O2/L. Observations of the effects of the test substance on time to hatch, hatching success, growth, and survival were used to calculate the NOEC and LOEC.

All viable embryos in the control and exposure replicates hatched by Days 6 or 7 of the test, and larvae were released into the test chambers on Day 6 of the test when > 90% of the viable embryos in the negative control replicates hatched.Daily observations of all the embryos indicated that there were no apparent differences in time to hatch between the control group and any of the treatment groups. Hatching success in the negative control group was 98%. Hatching success in the treatment groups was 96 - 100%. Fisher’s Exact test showed that there were no statistically significant decreases in hatching success at any of the test substance treatment groups when compared to the negative control (p > 0.05). 

Larval survival in the negative control group at test termination was 94% and 94 to 98%in the treatment groups. Fisher’s Exact test showed that there were no statistically significant decreases in larval survival noted in any of the test substance treatment groups when compared to the negative control data (p > 0.05). The majority of the fish in the negative control and treatment groups appeared normal throughout the test, with occasional observations of small fish. Occasional observations of small, lethargic, lying on the bottom of the test chamber and/or morphologically deformity (curled) were made in the negative control as well as in the 0.34 to 5.6 mg/L treatment groups. The observations of small and/or lethargic fish in the cation treatment groups were generally restricted to a few individuals and were generally comparable to the negative control group. Therefore, none of the sublethal effects noted for the fish in the 0.34 to 5.6 mg/L treatment groups were considered to be treatment-related. However, the number of small fish noted in the 14 mg /L treatment group was higher than the negative control and the rest of the treatment concentrations.

Statistically significant reductions in mean total length and mean wet weight were noted among fish in the 14 mg/L treatment group in comparison to the negative control (Dunnett’s one-tailed test, p ≤ 0.05). No statistically significant reductions in mean dry weight were noted in any of the test substance treatment groups in comparison to the negative control (Dunnett’s one tailed test, p > 0.05). Consequently, the NOEC was 5.6 mg test substance/L and the LOEC was 14 mg test substance/L, based on growth.