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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 210, EPS OPPTS 850.1400 and in accordance with the Principles of Good laboratory Practices (GLP)
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
Remarks:
Periodic analyses of water for potential contaminants were not performed according to Good Laboratory Practice Standards, but were performed using a certified laboratory and standard US EPA analytical methods
Qualifier:
according to
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
Deviations:
no
Remarks:
same as above
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Analytical monitoring:
yes
Details on sampling:
Water samples were collected from one test chamber of each treatment and control group three 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, 21, 28 and 33 (test termination) to determine concentrations of the test substance in the test chambers. All samples were collected at mid-depth in the test chambers, placed in glass vials (completely filled with no headspace) and processed immediately for analysis or stored under refrigerated conditions until analysis. All vials were purged with nitrogen gas prior to the addition of sample solutions.
Vehicle:
no
Details on test solutions:
The reported water solubility of the test material 1,3- and 1,4-cyclohexanecarboxaldehyde at 20.1 ± 0.5°C was 166 mg/mL. However, during the functional water solubility trial prior to the range finding test precipitate was noted in the stock solution at nominal concentrations of 30 and 93.4 mg a.i./mL. Consequently, the decision was made that the stock solution would be prepared at a nominal concentration of 1000 mg a.i./L during the range-finding test as well as the definitive test to reduce potential precipitate formation in the stock solution.
In order to minimize the contact of test stock solution with air, the RO water, which was used for the preparation of stock solutions, was purged with nitrogen gas and the surface of the water was covered with a blanket of nitrogen gas. The flasks and stock bottle used in the stock preparation were also purged with nitrogen gas prior to the addition of the solution and the nitrogen blanket was added to cover the waters’ surface.
A single stock solution was prepared every 1 to 5 days during the test. At each preparation, two 2000-mL stock solutions were prepared at a nominal concentration of 1000 mg a.i./L in RO water that was purged with nitrogen gas. Two 2000-mL glass class A volumetric flasks were filled with nitrogen purged RO water, then 2.228 mL of the water was removed from each flask prior to the addition of 2.228 mL of test substance under the water’s surface using gas-tight syringe. The flasks were purged with nitrogen gas, covered with Parafilm™and sonicated for approximately 150 to 360 minutes to mix. At the end of the mixing, the stock solutions appeared clear and colorless with white precipitate throughout the water column and at the bottom of the flasks. The remaining particulates in the stock solutions were removed by filtration through a coarse filter paper. The two stock solutions were combined in a 4000-mL glass amber bottle that was purged with nitrogen gas. The combined stock solution was purged with nitrogen gas once more before the bottle was capped and placed on the test system for delivery or until delivery. The test stock was adjusted for the purity of the active ingredient in the test substance of 91.6% and the estimated density of 0.98 g/mL
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: fathead minnow
- Strain: Pimephales promelas
- Source: Fathead minnow embryos used in the test were supplied by Chesapeake Cultures, Inc., Hayes, Virginia and were received at Wildlife International on spawning substrates. Upon receipt, the embryos were removed from the spawning substrates and examined under a dissecting microscope to select healthy, viable specimens at approximately the same stage of development. Embryos collected for use in the test were from ten individual spawns and were <24 hours old when the test was initiated. To initiate the test, groups of 1 to 3 embryos were impartially distributed among incubation cups until each cup contained 20 embryos. One cup was placed in each treatment and control test chamber.
Newly-hatched larvae were fed live brine shrimp nauplii (Artemia sp.) three times per day during the first seven days of post-hatch. Thereafter, they were fed live brine shrimp nauplii three times per day on weekdays and at least two times per day on weekends. Brine shrimp nauplii were obtained by hatching cysts purchased from Brine Shrimp Direct, Ogden, Utah. Fish were not fed for approximately 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.
Biomass loading at the end of the test, based on the mean wet weight of the negative control group, was 0.047 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 0.34 g fish/L.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
33 d
Post exposure observation period:
not applicable
Hardness:
Hardness (mg/L as CaCO3) - (mean) - 139, (range) - 136 – 148
Test temperature:
25 ± 1°C.
pH:
pH - (mean) - 8.0, (range) - 7.9 - 8.1
Dissolved oxygen:
Dissolved oxygen concentrations remained ≥80% of saturation (6.5 mg/L).
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentrations selected for use in the study were 0.63, 1.3, 2.5, 5.0 and 10 mg a.i./L. The measured concentrations of samples collected to verify the diluter system prior to the test ranged from 72.8 to 87.6% of nominal concentrations. When the measured concentrations of test solution samples collected on Days 0, 7, 14, 21, 28 and 33 of the test were averaged for each treatment group, the mean measured test concentrations were 0.26, 0.49, 0.92, 2.2 and 5.7 mg a.i./L, which represented 41, 38, 37, 44 and 57% of nominal concentrations, respectively.
Details on test conditions:
TEST SYSTEM
- The toxicity test was conducted using an exposure system consisting of a continuous-flow diluter to deliver each concentration of the test substance and a negative control (dilution water) to test chambers. The fluid metering pumps (Fluid Metering, Inc., Syosset, New York) were used to deliver test substance stock solution to mixing chambers indiscriminately assigned to each treatment. The stock solution was diluted with well water in the mixing chambers in order to obtain the desired test concentrations prior to delivery to the test chambers. The flow of dilution water into each mixing chamber was controlled using rotameters. After mixing, the test solution in each mixing chamber was pumped into the appropriate replicate test chamber using a peristaltic pump (Cole-Parmer Instrument Company, Chicago, Illinois), and was adjusted to provide approximately 7.2 volume additions of test solution in each test chamber per day. Periodically during the test, all organisms were transferred to clean test chambers to prevent the buildup of bacterial/fungal growth.
The pumps used to deliver the stock and test solutions, and the rotameters used to control the flow of dilution water to the mixing chambers, were calibrated prior to the test and verified or recalibrated, if necessary, approximately weekly during the test. The proportion of the test water that was pumped into each replicate test chamber was checked prior to the test and approximately weekly during the test to ensure that the actual volume delivered was within ± 5 of the desired flow rate. Delivery of test solutions to the test chambers was initiated six days prior to the introduction of the test organisms in order to achieve equilibrium of the test substance in the test system. The general operation of the exposure system was checked visually at least two times per day during the test and at least once on the last day of the test.
The delivery system and the test chambers were placed in a temperature-controlled environmental chamber to maintain the target water temperature throughout the test period. Test chambers were 9-L glass aquaria filled with approximately 7 L of test water. The depth of the test water in a representative test chamber was approximately 14 cm. Test chambers were labeled with the project number, test concentration and replicate. Embryos were held in incubation cups constructed from glass cylinders approximately 50 mm in diameter with 425 μm nylon screen mesh attached to the bottom with silicone sealant. The cups were 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.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International site. The well water was passed through a sand filter to remove particles greater than approximately 25 μm, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 μm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.
- Total organic carbon: (mg C/L) - (mean) - < 1
- Specific conductance ((μS/cm) - (mean) - 357, (range) - 350 -369
- Alkalinity: (mg/L as caCO3) - (mean) - 175, (range) - 174 - 176
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod and Light intensity: Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a photoperiod of 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 was measured at the water surface of one representative test chamber at test initiation using a SPER Scientific Model 840006 light meter.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : During the first day of exposure, embryos were observed twice for mortality and 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 5 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 dea th of the embryo occurred. During the 28-day post-hatch exposure period, the larvae were observed daily to evaluate the numbers of mortalities and the numbers 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 that hatched successfully.
Post-hatch growth of the fathead 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 45 hours to obtain dry weight data

RANGE-FINDING STUDY
- Test concentrations: In the range-finding test, embryos <24 hours old were impartially distributed to incubation cups and exposed to a series of test concentrations of 0.081, 0.27, 0.90, 3.0, 10 mg a.i./L or a negative control. Each test group had two replicates test chambers each containing 25 embryos. Embryos were exposed under flow-through conditions for 19 days (5 days hatch period and 14 days posthatch).
Since the test material was reported to react with air (oxygen) and was unstable under direct sunlight, the reverse osmosis (RO) water used for the preparation of stock solutions was purged with nitrogen gas and the surface of the water was covered with a blanket of nitrogen gas. During each step of
the stock solution preparation, nitrogen gas was used to purge the solution and to blanket the solution’s surface to minimize the contact of the test solution with air. The flasks used in the stock preparation as well as the stock solution bottle were also purged with nitrogen gas prior to the addition of the solution. The highest test concentration was based upon the functional water solubility trial conducted prior to the range-finding test; the highest stock solution achievable in RO water was 1000 mg a.i./L nominal concentration. Stock solution was prepared daily during the test. At each preparation, three 1000-mL primary stock solutions at 1000 mg a.i./L were prepared. A calculated amount of test substance (1.114 mL) was dissolved in each of three 1000-mL glass class A volumetric flask to achieve the final volume of 1000 mL in RO water, sonicated for approximately 150 to 360 minutes, followed by filtration using a coarse filter paper to remove any remaining precipitate. The three 1000-mL stock solutions were combined and placed in an amber bottle. The bottle was purged with nitrogen gas, capped and placed in the diluter delivery system. All test solutions appeared clear and colorless at initiation and termination of the range-finding test.
During the range-finding test, samples of stock solution were collected on Days 0 (new solution), 1 (new and old stock solutions), 2 (old stock solution) and 5 (old stock solution) of the test. Test solutions samples also were collected on Days 0, 7 and 19 of the test. Samples were collected into 40-mL glass vials that were purged with nitrogen gas and completely filled to eliminate headspace in the sample vial. The samples were processed to confirm the concentrations of 1,3- and 1,4-cyclohexanecarboxaldehyde in the stock and test solutions.
After a 5-day embryo hatching period, the larvae were released into the test chambers, where
exposure continued during a 14-day post-hatch juvenile growth period. At the end of the 14-day growth period, measurements of wet weight for each treatment and control group were made. Mean percent hatching success in the negative control group and in the 0.081, 0.27, 0.90, 3.0 and 10 mg a.i./L treatment groups was 100, 100, 100, 100, 100 and 100%, respectively. Mean percent post-hatch survival in the negative control group and in the 0.081, 0.27, 0.90, 3.0 and 10 mg a.i./L treatment groups was 96, 96, 90, 78, 100 and 82%, respectively. The mean wet weight in the negative control group and in the 0.081, 0.27, 0.90, 3.0 and 10 mg a.i./L treatment groups was 20.2, 22.2, 24.0, 20.6, 21.1 and 19.2 mg, respectively.
Based on the results of the preliminary range-finding tests, no apparent toxic effects were noted in any of the 1,3- and 1,4-cyclohexanecarboxaldehyde in comparison to the negative control. Consequently, the nominal test concentrations of 0.63, 1.3, 2.5, 5.0 and 10 mg a.i./L were selected in consultation with the Sponsor for the definitive test.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
5.7 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
2.2 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
other: total length, wet and dry weights
Details on results:
Daily observations of the embryos indicated that there were no apparent differences in time to hatch between the control group and any of the 1,3- and 1,4-cyclohexanecarboxaldehyde treatment groups. All of the viable fathead minnow embryos in the control and treatment replicates hatched on Day 5 or Day 6 of the test. Since hatching reached >90% in the control groups on Day 5 of the test, the larvae were released to their respective test chambers on that day. Any embryos remaining after Day 5 of the test were kept in the egg cup until death occurred, or until hatching on Day 6 of the test, at which time the newly hatched larva was added to the larval growth chamber.Hatching success in the negative control, 0.26, 0.49, 0.92, 2.2 and 5.7 mg a.i./L treatment groups was 100, 96, 100, 99, 100 and 98%, respectively. Fisher’s Exact test indicated that there was no statistically significant difference in hatching success in any of the 1,3- and 1,4-cyclohexanecarboxaldehyde treatment groups in comparison to the negative controls (p > 0.05). Consequently, the NOEC for hatching success was 5.7 mg a.i./L and the LOEC was > 5.7 mg a.i./L.
Larval survival on Day 14 post-hatch in the negative control, 0.26, 0.49, 0.92, 2.2 and 5.7 mg a.i./L treatment groups was 94, 92, 91, 92, 86 and 91%, respectively. Fisher’s Exact test indicated there was no statistically significant difference in survival in any of the 1,3- and 1,4-cyclohexanecarboxaldehyde treatment groups in comparison to the negative control (p > 0.05). Consequently, the 14-day post-hatch threshold level of lethal effect was > 5.7 mg a.i./L. One fish in the 2.2 mg a.i./L treatment group was injured during transfer on Day 21 post-hatch (Day 26 of the test) and was later found dead. Since the mortality was not treatment related, it was excluded from the analysis of survival at test end. Larval survival in the negative control, 0.26, 0.49, 0.92, 2.2 and 5.7 mg a.i./L treatment groups was 94, 92, 91, 91, 86 and 88%, respectively. Fisher’s Exact test indicated there was no statistically significant difference in survival in any of the 1,3- and 1,4-cyclohexanecarboxaldehyde treatment groups in comparison to the negative control (p > 0.05). Consequently, the NOEC for larval survival was 5.7 mg a.i./L and the LOEC was > 5.7 mg a.i./L.
The majority of the larvae appeared normal during the 14 Day post-hatch period, with one or two observations of small, discoloration, weak and/or morphological deformity (i.e. curled/curved spine or tail). The numbers of deformed and small larvae noted at Day 14 post-hatch were infrequent and were comparable among fish in the treatment groups. These observations were considered to be natural occurrence in the population. Therefore, the statistical analyses of these endpoints were not performed. Consequently, the 14-day post-hatch threshold level of observed effects was >5.7 mg a.i./L.
In general, the majority of the fish in the control groups and in the 0.26, 0.49, 0.92, 2.2 and 5.7 mg a.i./L treatment groups appeared normal throughout the test. There were infrequent observations of organisms that were noted with sublethal effect or abnormal behavior. The observations of sublethal effects include weak, pale, smaller fish in comparison to the fish in the control replicates, morphological deformity (i.e. curled/curved spine or tail, small pupil), and redness around the abdominal area (possibly due to hemorrhage). Some of the abnormal behaviors noted were erratic swimming, lying at the bottom of the test chamber or surfacing. These observations in the 0.26 to 2.2 mg a.i./L treatment groups were infrequent. However, the frequency of the sublethal effects increased substantially in the 5.7 mg a.i./L treatment group, which deemed the effects treatment related.
Dunnett’s one-tailed test indicated there was a statistically significant reduction in total length among fish in the 0.26 and 5.7 mg a.i./L treatment groupswhen compared to the negative control (p ≤ 0.05). However, the decrease in total length at the 0.26 mg a.i./L treatment group did not follow a dose-response pattern, was not reflected in significant reductions in weight and was not considered to be biologically meaningful. Significant reductions in wet weight and dry weight were noted among fish in the 5.7 mg a.i./L treatment group in comparison to the negative control (p ≤ 0.05). Consequently, the NOEC for growth was 2.2 mg a.i./L and the LOEC was 5.7 mg a.i./L.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Standard statistical methods were employed

None

Validity criteria fulfilled:
yes
Conclusions:
Fathead minnows (Pimephales promelas) were exposed to 1,3- and 1,4-cyclohexanecarboxaldehyde at mean measured concentrations of 0.26 to 5.7 mg a.i./L under flow-through conditions for 33 days (a 5-day hatching period plus a 28-day post-hatch growth period). There were no statistically significant treatment-related effects on hatching success or survival at concentrations ≤5.7 mg a.i./L. Growth, measured as total length, wet and dry weight, was the most sensitive biological endpoint measured in this study. Fathead minnows exposed to 1,3- and 1,4-cyclohexanecarboxaldehyde at concentrations 5.7 mg a.i./L had statistically significant reductions in total length, wet weight and dry weight in comparison to the negative control. Consequently, the NOEC, based on growth, was 2.2 mg a.i./L. The LOEC was 5.7 mg a.i./L and the MATC was calculated to be 3.5 mg a.i./L.
The 14-day post-hatch threshold levels of lethal effect were assessed based on the 14-day posthatch data on percent mortality and clinical observations and were both determined to be > 5.7 mg a.i/L. The 14-day post-hatch NOEC was estimated to be 5.7 mg a.i./L.
Executive summary:

Fathead minnow embryos were exposed to a geometric series of five test concentrations and a negative (dilution water) control under flow-through conditions. The exposure period included a 5-day embryo hatching period, and a 28-day post-hatch juvenile growth period. Nominal test concentrations were 0.63, 1.3, 2.5, 5.0 and 10 mg active ingredient (a.i.)/L. The concentrations were selected in consultation with the Sponsor, and were based on exploratory range-finding toxicity data (Table 1). Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at the beginning of the test, at weekly intervals during the test and at test termination.

Delivery of the test solutions to the test chambers was initiated six days prior to test initiation in order to achieve equilibrium of the test substance. Four replicate test chambers were maintained in each treatment and control group, with one incubation cup in each test chamber. Each incubation cup contained 20 embryos, resulting in a total of 80 embryos per treatment. At test initiation, embryos <24 hours old were impartially distributed to incubation cups and exposed to test solution in the test chambers. After a 5-day embryo hatching period, the larvae were released into the test chambers, where exposure continued during a 28-day post-hatch juvenile growth period. Observations of the effects of 1,3- and 1,4-cyclohexancarboxaldehydeon time to hatch, hatching success, growth, and survival were used to calculate the no-observed-effect-concentration (NOEC), the lowest-observed-effect-concentration (LOEC), and the maximum acceptable toxicant concentration (MATC).

Fathead minnows (Pimephales promelas) were exposed to 1,3- and 1,4-cyclohexanecarboxaldehyde at mean measured concentrations of 0.26 to 5.7 mg a.i./L under flow-through conditions for 33 days (a 5-day hatching period plus a 28-day post-hatch growth period). There were no statistically significant treatment-related effects on hatching success or survival at concentrations ≤5.7 mg a.i./L. Growth, measured as total length, wet and dry weight, was the most sensitive biological endpoint measured in this study. Fathead minnows exposed to 1,3- and 1,4-cyclohexanecarboxaldehyde at concentrations 5.7 mg a.i./L had statistically significant reductions in total length, wet weight and dry weight in comparison to the negative control. Consequently, the NOEC, based on growth, was 2.2 mg a.i./L. The LOEC was 5.7 mg a.i./L and the MATC was calculated to be 3.5 mg a.i./L.

The 14-day post-hatch threshold levels of lethal effect were assessed based on the 14-day posthatch data on percent mortality and clinical observations and were both determined to be > 5.7 mg a.i/L. The 14-day post-hatch NOEC was estimated to be 5.7 mg a.i./L.

Description of key information

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
1.68 mg/L

Additional information

Fathead minnow embryos were exposed to a geometric series of five test concentrations and a negative (dilution water) control under flow-through conditions. The exposure period included a 5-day embryo hatching period, and a 28-day post-hatch juvenile growth period. Nominal test concentrations were 0.63, 1.3, 2.5, 5.0 and 10 mg active ingredient (a.i.)/L. The concentrations were selected in consultation with the Sponsor, and were based on exploratory range-finding toxicity data. Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at the beginning of the test, at weekly intervals during the test and at test termination.

Delivery of the test solutions to the test chambers was initiated six days prior to test initiation in order to achieve equilibrium of the test substance. Four replicate test chambers were maintained in each treatment and control group, with one incubation cup in each test chamber. Each incubation cup contained 20 embryos, resulting in a total of 80 embryos per treatment. At test initiation, embryos <24 hours old were impartially distributed to incubation cups and exposed to test solution in the test chambers. After a 5-day embryo hatching period, the larvae were released into the test chambers, where exposure continued during a 28-day post-hatch juvenile growth period. Observations of the effects of 1,3- and 1,4-cyclohexancarboxaldehydeon time to hatch, hatching success, growth, and survival were used to calculate the no-observed-effect-concentration (NOEC), the lowest-observed-effect-concentration (LOEC), and the maximum acceptable toxicant concentration (MATC).

Fathead minnows (Pimephales promelas) were exposed to 1,3- and 1,4-cyclohexanecarboxaldehyde at mean measured concentrations of 0.26 to 5.7 mg a.i./L under flow-through conditions for 33 days (a 5-day hatching period plus a 28-day post-hatch growth period). There were no statistically significant treatment-related effects on hatching success or survival at concentrations5.7 mg a.i./L. Growth, measured as total length, wet and dry weight, was the most sensitive biological endpoint measured in this study. Fathead minnows exposed to 1,3- and 1,4-cyclohexanecarboxaldehyde at concentrations 5.7 mg a.i./L had statistically significant reductions in total length, wet weight and dry weight in comparison to the negative control. Consequently, the NOEC, based on growth, was 2.2 mg a.i./L. The LOEC was 5.7 mg a.i./L and the MATC was calculated to be 3.5 mg a.i./L.

The 14-day post-hatch threshold levels of lethal effect were assessed based on the 14-day post-hatch data on percent mortality and clinical observations and were both determined to be > 5.7 mg a.i/L. The 14-day post-hatch NOEC was estimated to be 5.7 mg a.i./L.

 

 

 

According to "OECD Guidelines for the Testing of Chemicals 215 Fish, Juvenile Growth Test, 2000", the effect on growth of juvenile fish from exposure of the test substance UNOXOLTM 3,4-DIALDEHYDE (DOWM20140430-0892) to Chinese Rare Minnow (Gobiocypris rarus) was determined by a flow-through test. The test substance was dissolved in the test water stirred (via magnetic stirrer) in the dark for 10 min at 500 rpm and filtered through a 0.45 pm nitrocellulose membrane to prepare a stock solution with a concentration of 500 mg/L (the average measured concentration was 425 mg/L in the test). The test solutions were prepared by dilution of the stock solution with test water. The stock solution was renewed daily. Based on the results of the fish acute toxicity test, five treatment groups and a control group were used in the test. The nominal concentrations were 0.625, 1.25, 2.50, 5.00 and 10.0 mg/L (their arithmetic mean measured concentrations were 0.627, 1.24, 2.47, 4.75 and 9.85 mg/L, respectively). A peristaltic pump system continuously dispensed the stock solutions and test water to the mixing chambers. The frequency of water removal at the beginning of test was 6 liters/g of fish/day. The test solutions flowed through each test chamber at the flow-rate of 6.72 mL/min and 9.68 liters of test solution flowed through each test chamber per 24 hours. All groups had no replicate with twenty fish each. The test duration was 28 days.

The mortality in the control group was zero at the end of the test and the dissolved oxygen concentration was greater than 60.0% of the air saturation value (ASV) throughout the test. The temperature varied less than ±1°C and the mean weight of fish in the control increased by 107%, more than the half of their mean initial weight over 28 days. Hence, the test was considered to be valid. The concentrations of the test substance in test solutions were analyzed using a HPLC (High Performance Liquid Chromatography) method. In the test, the measured concentrations of test substance in the test solutions varied within ±20% of their nominal concentrations (- 14.9%--14.6%). The test results were expressed relative to the arithmetic mean measured concentrations. Under tested conditions, the EC10 and EC20 values of growth rate inhibition for Gobiocypris rarus exposed to the test substance for 28 days was 1.68 mg/L and 7.20 mg/L, respectively.