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Long-term toxicity to fish

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Endpoint:
long-term toxicity to fish, other
Remarks:
Fish prolonged toxicity test
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling: Samples of the test solutions were taken for analysis on days 1, 2, 3, 6, 11, 13, 19, 21, 25 and 27. The samples were taken from the centre of each test solution.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- A primary 10 g/L stock solution was prepared by dissolving 0.7 g of the test substance in 70 mL of dimethylformide (DMF) and stirring thoroughly. Stock solutions (each nominally 10000 x the strength of the respective test solution) were prepared by measuring into glass jars the amount of primary 10 g/L stock solution required and making up to a nominal 300 mL with DMF. The stock solutions were then stirred to ensure complete mixing. The stock solution for the solvent control consisted of 300 mL of DMF alone. Stock solutions were replaced on days 7, 14 and 21. After dilution all test concentrations and the solvent control contained a nominal 100 µL of DMF per litre.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss
- Source: Houghton Springs Fish Farm, Winterbourne, Houghton, UK
- Length at test initiation: 56 mm (mean)
- Length at end of exposure period: 66 - 81 mm (mean: 74 mm)
- Weight at end of exposure period: 5.06 - 8.48 g (mean: 6.99 g)
- Feeding during test: Fish were fed with Promin (a proprietary product) at a rate of 2% of the total body weight of fish present per day

ACCLIMATION
- Acclimation period: 22 days
- Type and amount of food during acclimation: Fish were fed with Promin (a proprietary product) daily
- Health during acclimation: The fish showed no evidence of disease during holding; therefore no medication was necessary.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
28 d
Hardness:
42.3 - 54.0 mg CaCO3/L (mean: 47.6 mg CaCO3/L)
Test temperature:
14.8 - 15.2°C (mean: 15.0°C)
pH:
7.20 - 7.95
Dissolved oxygen:
8.4 - 9.8 mg/L (mean: 9.1 mg/L)
Conductivity:
254 μS/cm (228 - 283 μS/cm)
Nominal and measured concentrations:
Nominal: 10, 18, 32, 56 and 100 μg/L
Mean measured: 11, 19, 34, 36 and 100 μg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: The apparatus used in this study was a dynamic, continuous flowthrough system. The test vessels, dosing lines, mixing chambers and stock vessels were all constructed of glass. A minimum quantity of silicone rubber tubing was used to connect the components together.
Glass vessels (610 x 305 x 310 mm; length x width x depth) with a maximum capacity of 54 litres and a working volume of 45 litres were used to hold the test fish. The test vessels had loose fitting glass covers.
- Aeration: No
- No. of organisms per concentration: 10
- Type of flow-through: dynamic, continuous
- Renewal rate of test solution: The test solutions were renewed at a nominal rate of 250 mL/min so that a nominal 95% exchange of the test solutions was calculated to occur within 9 hours.
- No. of vessels per concentration (replicates): No replicates
- No. of vessels per control (replicates): No replicates
- No. of vessels per vehicle control (replicates): No replicates
- Loading of dilution water control fish: 1.6 g/L


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was dechlorinated tap water supplied from a 100 m³ reservoir with an average retention time of 24 hours. It was passed through activated carbon, coarsely filtered to remove particulate material and dechlorinated with sodium thiosulphate. The treated water was then held in a secondary reservoir with a capacity of 36 m³ and an average retention time of 8 hours. The water was then passed through an ultra violet sterilizer to a second set of filters of 25 and 10 μm and then to a third storage tank with a capacity of 13.5 m³. The treated water was then delivered via a ring circuit to a temperature controlled header tank in the test laboratory set to a nominal temperature of 15°C and finally filtered to 5 μm before use.
- Alkalinity: 35.9 mg CaCO3/L
- Chlorine: <4 μg/L

OTHER TEST CONDITIONS
- Photoperiod: 16 hr photoperiod and 8 hr darkness with 10 min transition period

EFFECT PARAMETERS MEASURED: Observations for mortalities and symptoms of toxicity were made daily.
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
27 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CI: 20 - 36 µg/L
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
10 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Mortality and symptoms of toxicity
Details on results:
- Concentrations that produce lethal or other effects: The lowest nominal concentration at which there was 100% mortality was 56 µg/L. Individual fish in the 32 µg/L test concentration were observed to be distressed and were terminated to prevent unnecessary suffering on days 12, 13, 20 and 22. Similarly one fish in the 18 µg/L test concentration was terminated on day 25.
- Cumulative mortality at each concentration and for each recommended observation time if possible: After 28 days, mortality was 0, 0, 0, 10, 60, 100, and 100% in the 0 (control), 0 (solvent control), 10, 18, 32, 56 and 100 μg/L treatments, respectively
- Mortality in the controls: None
- Behavioural observation of the fish: The general symptoms of toxicity noted in this study were surfacing, dark discolouration, sounding, loss of balance, reduced feeding, quiescence, haemorrhaging, skittering, gulping air, damaged fins, twitching, cataracts and irregular and rapid respiration.
Conclusions:
28 d LC50: 27 µg/L (mortality) (95% CI: 20 - 36 µg/L)
28 d NOEC: 10 µg/L (mortality and symptoms of toxicity)
Executive summary:

The toxicity of the test substance to the Rainbow trout (Oncorhynchus mykiss), was determined in a 28 day flowthrough test according to OECD guideline 204.

The study was conducted with five nominal concentrations of the test substance (10, 18, 32, 56 and 100 μg/L), a dilution water control, and a solvent control at a temperature range of 14.8 - 15.2°C.

Mean measured concentrations of the test substance during the exposure were <LOD (control), <LOD (vehicle), 11, 19, 34, 36 and 100 μg/L.

After 28 days, mortality was 0, 0, 0, 10, 60, 100, and 100% in the 0 (control), 0 (solvent control), 10, 18, 32, 56 and 100 μg/L treatments, respectively. No mortality were observed in the dilution water control or solvent control. The nominal threshold level of lethal effect was determined at 18 µg/L and the nominal threshold level of observed effect was determined at 18 µg/L.

The lowest nominal concentration at which there was 100% mortality was 56 μg/L. The 28 day nominal no observed effect concentration (NOEC) of the test substance, based on symptoms of toxicity, was 10 μg/L. The 28 day LC50 was 27 μg/L.

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: One replicate of each treatment level and the controls were sampled prior to the start of the definitive exposure.
During the in-life phase of the definitive study, samples were removed from alternating replicate solutions of each treatment level and control on days 0, 5, 12, 19, 26, and 33 (day 28 post-hatch)
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Preparation of stock solution: A 5.0 mg a.s./mL diluter stock solution was prepared by placing approximately 0.2518 g of the test substance (0.2500 g a.s., adjusted for purity) in a 50-mL volumetric flask and bringing it to volume with DMF. The resulting stock solution was observed to be clear and colorless.
A 120 μL/mL solvent control stock solution was prepared by bringing 120 mL of DMF to a total volume of 1000 mL with deionized water. The resultant stock solution was observed to be clear and colorless.
- Differential loading: Prior to test initiation, a Harvard Apparatus Syringe Pump in conjunction with a 50-mL Glenco® gas-tight syringe was calibrated to deliver 0.0388 mL/cycle of the 5.0 mg a.s./mL stock solution into the diluter system's chemical mixing chamber, which received 1.94 L/cycle of dilution water. The mixing chamber was positioned in an ultrasonic water bath containing a water-driven stirrer which aided in the solubilization of the test substance into the dilution water. The concentration of the test substance in the solution contained within the mixing chamber was equivalent to that of the highest nominal test concentration (100 μg a.s./L) and was proportionally diluted (50%) to produce the remaining nominal test concentrations (50, 25, 13, and 6.3 μg a.s./L).
- Controls: dilution water control, and a solvent control
- Chemical name of vehicle: dimethylformamide (DMF)
- Concentration of vehicle in test medium: 20 μL/L
Test organisms (species):
Cyprinodon variegatus
Details on test organisms:
TEST ORGANISM
- Common name: sheepshead minnow
- Strain: Cyprinodon variegatus
- Source: Aquatic Biosystems, Fort Collins, Colorado

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Twenty-eight labeled incubation egg cups containing control water were impartially placed in a water bath maintained at 25ºC. A glass bowl containing the embryos was placed in the same water bath. The embryos were impartially placed in the embryo incubation cups five at a time until each cup contained five eggs. This process was repeated until all incubation cups contained 30 eggs. Once all incubation cups contained 30 embryos, the embryos in each cup were microscopically examined for viability; non-viable embryos were removed and replaced. To initiate the study, the incubation cups, each containing 30 embryos, were then suspended in the respective exposure aquaria (one cup per replicate vessel). At study initiation, the embryos were less than 30 hours old.

POST-HATCH FEEDING
- Start date: day 5 (day 0 post-hatch)
- Type/source of feed: brine shrimp nauplii (Artemia salina)
- Amount given: ad libitum
- Frequency of feeding: three times daily
Larvae were not fed during the 24 hours prior to study termination
Test type:
flow-through
Water media type:
saltwater
Limit test:
yes
Total exposure duration:
28 d
Remarks on exposure duration:
Length of test: 28 days post-hatch (33 days overall)
Test temperature:
24.0-28.0°C
pH:
7.6-8.1
Dissolved oxygen:
greater than 60% saturation
Salinity:
19-21‰
Nominal and measured concentrations:
Nominal: 6.3, 13, 25, 50 and 100 μg a.s./L
Measured: 5.2, 11, 21, 49, and 100 μg a.s./L
Details on test conditions:
TEST SYSTEM
- Emybro cups: Embryo incubation cups were round glass jars (5 cm) diameter, 8 cm high with 475-μm nylon screen bottoms.
- Test vessel, Material, size, headspace, fill volume: The diluter system and exposure aquaria were fabricated of glass and silicone sealant. Each test aquarium measured 30 x 15 x 20 cm with a 14.5-cm high side drain that maintained a constant exposure solution volume of 6.5 L.
- Type of flow-through: intermittent-flow proportional diluter
- No. of fertilized eggs/embryos per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: During the 28-day post-hatch exposure period, biomass loading did not exceed 0.054 g/L of flowing test solution per day in any replicate exposure aquarium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural, filtered seawater from the Cape Cod Canal, Bourne, Massachusetts from about 1 to 4 meters offshore at a depth of approximately 0.5 meters. In the laboratory, the seawater was adjusted to a salinity of 20 ± 3‰ with laboratory well water, filtered through 20- and 5-μm polypropylene core filters and recirculated within an epoxy-coated concrete holding reservoir prior to use.
- Chlorine: less than detection limit
- Salinity: 19 to 21‰

OTHER TEST CONDITIONS
- Photoperiod: 16 hr light and 8 hr dark including a 30 min transitional period preceding and following the 16-hr light interval.
- Light intensity: 630 to 1290 lux at test termination

EFFECT PARAMETERS MEASURED: The behavior and appearance of larvae were observed and recorded daily. Larval survival was also evaluated daily. At 28 days post-hatch exposure (test termination), the percentage of larval survival was determined. The surviving larvae were euthanized and each larval fish in each exposure aquarium was then measured individually to determine the total length. The fish were then dried in an oven at 95 to 102ºC for 23 hours and their individual dry weights were measured.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 6.3, 13, 25, 50, and 100 μg a.s./L , a dilution water (blank) control and a solvent (DMF) control
- Results used to determine the conditions for the definitive study: Yes

POST-HATCH DETAILS
- Begin of post-hatch period: test day 5
- No. of hatched eggs (alevins)/treatment released to the test chamber: On test day 5, the surviving larvae present in each incubation cup were randomly thinned to 10 organisms per replicate/40 organisms per treatment level or control and placed into their respective exposure aquaria.

FERTILIZATION SUCCESS STUDY
Calculation of percent hatching success was based on the number of live, dead, and deformed larvae per incubation cup after hatching was completed (day 5) compared to the number of embryos per cup on test day 0.
Reference substance (positive control):
no
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
21 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks:
total length and dry weight
Key result
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
49 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks:
total length and dry weight
Duration:
33 d
Dose descriptor:
other: MATC
Effect conc.:
32 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks:
total length and dry weight
Details on results:
- Mortality/survival at embryo, larval, juvenile, and adult stages: Following 28-days post-hatch exposure (day 33), larval survival in the control and solvent control averaged 98 and 95%, respectively. Mean larval survival in the 5.2, 11, 21, and 49 μg a.s./L mean, measured treatment levels was 90, 98, 95, and 93%, respectively.
- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: At the completion of the hatching period (day 5), embryo hatching success in the control and solvent control averaged 98 and 100%, respectively. Mean percent hatching success in the 5.2, 11, 21, 49, and 100 μg a.s./L mean, measured treatment levels was 98, 100, 99, 100, and 97%, respectively.
- Observations on body length and weight of young and/or exposed parents at one or more time periods: At test termination, the average total length of larvae exposed to the control and solvent control was 26.5 and 26.6 mm, respectively. Mean total length of larvae in the 5.2, 11, 21, and 49 μg a.s./L mean, measured treatment levels was 26.6, 26.0, 26.2, and 25.0 mm, respectively.
At test termination, the average dry weight of the test organisms in the control and solvent control was 0.0692 and 0.0724 g, respectively. Mean dry weight of larvae exposed to the 5.2, 11, 21, and 49 μg a.s./L mean, measured treatment levels was 0.0727, 0.0682, 0.0686, and 0.0624 g, respectively.
- Number of healthy fish at end of test: The number of healthy larvae exposed to the control and solvent control both averaged 10 larvae. The number of healthy larvae exposed to mean, measured concentrations of 5.2, 11, 21, and 49 μg a.s./L averaged 9, 10, 10, and 9 larvae, respectively.
Reported statistics and error estimates:
Analyses were performed using the mean organism response in each treatment group rather than individual response values. All statistical analyses were conducted at the 95% level of certainty except in the case of Shapiro-Wilks' and Bartlett's Tests, in which the 99% level of certainty was applied. The 99% level of certainty is preferred for qualifying tests. The following procedures were used:
1. Student’s t-Test was used to evaluate the endpoints and to compare the performance of the dilution water (blank) control organisms with that of the solvent control organisms. No significant difference was determined, therefore, the data were pooled for comparison with the treatment data.
2. Significant differences in embryo hatching success and the percentage survival were determined after arcsine square-root percentage transformation of the data.
3. Shapiro-Wilks' Test for normality was used to compare the observed sample distribution with a normal distribution for all endpoints. For this study, data for all endpoints were normally distributed with the exception of healthy larvae at test termination data.
4. As a check on the assumption of homogeneity of variance, implicit in parametric statistics, data for each endpoint were analyzed using Levene's Test. For this study, data for all endpoints met the homogeneity of variance assumption.
5. For this study, all endpoints with the exceptions of embryo hatching success and healthy larvae at test termination met the assumptions for normality and homogeneity of variance. Therefore, the performance of organisms exposed to each treatment level was compared with the pooled control data using Williams' Test to determine treatment-related effects. Healthy larvae at test termination data did not meet the assumption for homogeneity of variance and were evaluated using Dunn’s Multiple Comparison Test.
TOXSTAT version 3.5 (West, Inc. and Gulley, 1996) was used to perform the statistical computations.
Validity criteria fulfilled:
yes
Conclusions:
33 d NOEC: 21 µg/L (total length and dry weight)
33 d LOEC: 49 µg/L (total length and dry weight)

Executive summary:

The early life-stage toxicity of the test substance to sheepshead minnow, Cyprinodon variegatus, under flow-through conditions was determined in a 33-day exposure test. The test was conducted in accordance with the OECD Guideline for the Testing of Chemicals Number 210, Fish, Early Life-Stage Toxicity Test (OECD, 1992) and the U.S. Environmental Protection Agency’s Ecological Effect Test Guideline OPPTS Ecological Effects Test Guidelines (draft) 850.1400, Fish Early Life-Stage Toxicity Test (U.S. EPA, 1996).

The study was conducted with five concentrations of the test substance, a dilution water control, and a solvent (dimethylformamide, DMF) control at a temperature range of 24 to 28°C. Four replicate aquaria were initiated for each test substance concentration and control. Eggs were impartially distributed, five at a time, to each of 28 labeled egg cups (one egg cup per aquaria; 4 cups per test concentration) using a serological pipette. The process was repeated until each cup contained 30 eggs.

Mean, measured concentrations of the test substance ranged from 82 to 100% of the nominal the test substance concentrations during the test. At the completion of the hatching period (day 5), embryo hatching success in the control and solvent control averaged 98 and 100%, respectively. Embryo hatching success at mean, measured test substance concentrations of 5.2, 11, 21, 49, and 100 μg a.s./L averaged 98, 100, 99, 100, and 97%, respectively. Williams’ Test determined a significant difference in embryo hatching success at a mean, measured concentration of 100 μg a.s./L compared to the pooled control. Therefore, the No-Observed-Effect Concentration (NOEC) for hatching success was 49 μg a.s./L.

At the completion of the hatching period, there were no surviving larvae exposed to the 100 μg a.s./L mean, measured treatment level; therefore, no surviving larvae were continued for the 28-day post-hatch exposure. This treatment level was excluded from further statistical analysis.

The percent of normal larvae in the control and solvent control were both 100%. Percent of normal larvae at mean, measured test substance concentrations of 5.2, 11, 21, and 49 μg a.s./L were 100, 100, 100, and 100%, respectively. Since no abnormal larvae were observed at hatch in any treatment level or control, this data set was not statistically analyzed.

Following 28 days post-hatch exposure (day 33), larval survival among control and solvent control organisms averaged 98 and 95%, respectively. Larval survival at mean, measured test substance concentrations of 5.2, 11, 21, and 49 μg a.s./L averaged 90, 98, 95, and 93 respectively. Williams’ Test determined no significant difference in larval survival in any treatment level compared to the pooled control. Therefore, the No-Observed-Effect Concentration (NOEC) for larval survival was 49 μg a.s./L. The number of healthy larvae exposed to the control and solvent control both averaged 10 larvae.

The number of healthy larvae exposed to mean, measured test substance concentrations of 5.2, 11, 21, and 49 μg a.s./L averaged 9, 10, 10, and 9 larvae, respectively. Dunns' Multiple Comparison Test determined no significant difference in number of healthy larvae in any treatment level compared to the pooled control. Therefore, the No-Observed-Effect Concentration (NOEC) for healthy larvae was 49 μg a.s./L.

At test termination, average total length of larvae exposed to the control and solvent control was 26.5 and 26.6 mm, respectively. Total length of larvae exposed to mean, measured test substance concentrations of 5.2, 11, 21, and 49 μg a.s./L averaged 26.6, 26.0, 26.2, and 25.0 mm, respectively. Williams' Test determined a significant difference in total length among organism exposed to the 49 μg a.s./L treatment level compared to the pooled control. Therefore,the No-Observed-Effect Concentration (NOEC) for average total length was 21 μg a.s./L.

At test termination, average dry weight of larvae exposed to the control and solvent control was 0.0692 and 0.0724 g, respectively. Dry weight of larvae exposed to mean, measured test substance concentrations of 5.2, 11, 21, and 49 μg a.s./L averaged 0.0727, 0.0682, 0.0686, and 0.0624 g, respectively. Williams' Test determined a significant difference in dry weight among organisms exposed to the 49 μg a.s./L treatment level compared to the pooled control.

Therefore, the No-Observed-Effect Concentration (NOEC) for average dry weight was 21 μg a.s./L.

Based on growth (total length and dry weight) and mean, measured concentrations of the test substance, the NOEC was 21 μg a.s./L. The Lowest-Observed-Effect Concentration (LOEC) and the Maximum-Allowable-Toxicant Concentration (MATC), based on mean, measured concentrations of the test substance and growth, were 49 and 32 μg a.s./L, respectively.

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: US Environmental Protection Agency guideline EPA 540/9-86-138
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling: Samples of one replicate for each test concentration were taken on days 0 and 2 and then once per week until the end of the study. During the study sampling was alternated between all four replicates. On each of these sampling occasions one test concentration was sampled in triplicate. On days 1, 5, 10 and 20 four test concentrations were sampled in all four test replicates (including the dilution water and solvent controls). For one of the test concentrations sampled on these days one replicate was also sampled in triplicate.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- A 0.80 g/L stock concentrate was prepared by dissolving 0.56 g of the test substance in 700 mL of triethylene glycol followed by 5 minutes ultrasonic treatment and 5 minutes stirring, resulting in a clear, pale yellow solution. From exposure day 100.32 g of the test substance were added to 400 mL triethylene glycol and treated in the same manner.
A series of stock solutions was prepared by addition of the required volume of stock concentrate to 500 mL glass jars.
A magnetic follower was added to each vessel and the contents stirred for approximately 60 seconds on a magnetic stirrer.
The stock solution vessels were then weighed and placed on the test apparatus on magnetic stirrers. These stock solutions were continuously stirred during the test and were delivered at a nominal flow rate of 0.025 mL/min to the mixing chambers. The flow rates of the stock solutions and of the dilution water were measured on day 0 and thereafter three times per week. The nominal dilution achieved at this stage, immediately before delivery to the exposure concentrations and solvent control test tanks, was 10000 times. The stock solutions were replenished at weekly intervals (on exposure days -4, 3, 10, 17, 24 and 31).
- Controls: Dilution water control and solvent (triethylene glycol) control
- Chemical name of vehicle: triethylene glycol
- Concentration of vehicle in test medium: The solvent control and each concentration of the test substance contained a constant nominal 100 µL/L of triethylene glycol
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Strain: Pimephales promelas
- Source: Multi aquaculture Systems Inc., PO Box 679, Amagansett, New York, 11930, U.S.A.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish: 5 - 10 fish in each tank
- Collection and handling of fertilised eggs: The eggs from the spawnings in 9 brood tanks were collected and pooled in a dish filled with dilution water. Each batch of eggs was less than 24 hours old, that is up to and including the tail-bud stage. Sets of five eggs were randomly selected, microscopically examined for viability and placed in incubation cups. This process was repeated until each incubation cup contained a nominal 20 randomly selected eggs.

POST-HATCH FEEDING
- The larvae were fed three times a day during the week (Monday to Friday) and twice a day at weekends. From post hatch day 1 (exposure day 5) until post hatch day 4 the larvae were fed on freshwater rotifers (Brachionus plicatitus) at a rate of 3 ml rotifer culture per replicate tank per feed. The 80 µg/L tanks were not fed for one of the feeds on post hatch day 1 due to the hatch not being complete. On post hatch day 4 the larvae were also fed brine shrimp (Artemia salina) at a rate of 0.67 ml brine shrimp culture per fry per feed. On post hatch day 5 the larvae were fed brine shrimp only, at a rate of 0.67 ml brine shrimp culture per fry per feed. On post hatch day 6 and 7 the larvae were fed at a rate of 1.33 mL brine shrimp culture per fry per feed. From post hatch day 8 until post hatch day 15 the larvae were fed brine shrimp culture at a rate of 2.0 ml per fry per feed. From post hatch day 16 the larvae were fed brine shrimp culture at a rate of 3.0 mL per fry per feed and one of the brine shrimp feeds was replaced with a high protein pelleted fish food. The larvae were not fed during the last two days of the study.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
36 d
Remarks on exposure duration:
Length of test: 32 days post-hatch (36 days overall)
Hardness:
18.6 - 57.6 mg CaCO3/L
Test temperature:
24.0 - 25.3°C
pH:
7.42 - 8.19
Dissolved oxygen:
6.6 - 8.2 mg/L
Salinity:
<1 - 1.5‰
Conductivity:
130 - 274 μS/cm
Nominal and measured concentrations:
Nominal: 5, 10, 20, 30, 40 and 80 μg/L
Measured: 4.8, 10, 19, 27, 36 and 73 μg/L
Details on test conditions:
TEST SYSTEM
- Emybro cups: Egg incubation cups were made from 80 mm lengths of 50 mm diameter glass tubing with nylon mesh (0.47 mm²) attached to the bottom of each cup using silicone sealant. The cups were suspended in the test vessels and oscillated vertically over a distance of approximately 20-50 mm at a rate of 2 oscillations/min. Each replicate test vessel contained one incubation cup.
- Test vessel, Material, size, headspace, fill volume: The test apparatus was constructed of glass with a minimum of other materials (PVC tubing and sealant) in contact with the test solutions. The test vessels were of all glass construction, rectangular in shape with dimensions of 305 x 205 x 210 mm (length x width x height) and a maximum capacity of approximately 12 L. The test solution volume used was nominally 9.5 L and the water depth in each vessel was approximately 150 mm.
- Aeration: No
- No. of organisms per concentration: 10
- Type of flow-through: dynamic, continuous
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Loading: 2.0 eggs per litre of test solution on a static basis and a nominal flow loading of 120 ml per egg per hour.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was dechlorinated tap water supplied from a 100 m³ reservoir with an average retention time of 24 hours. It was passed through activated carbon, coarsely filtered to remove particulate material and dechlorinated with sodium thiosulphate. The treated water was then held in a secondary reservoir with a capacity of 36 m³ and an average retention time of 8 hours. The water was then passed through an ultra violet sterilizer to a second set of filters of 25 and 10 μm and then to a third storage tank with a capacity of 13.5 m³. The treated water was then delivered via a ring circuit to a temperature controlled header tank in the test laboratory set to a nominal temperature of 15 ± 1°C and finally re-filtered to 5 μm before use.
- Alkalinity: 15.0 - 38.6 mg CaCO3/L
- Chlorine: < 4 μg/L

OTHER TEST CONDITIONS
- Photoperiod: 16 hr photoperiod and 8 hr darkness with 20 min transition period
- Light intensity: 480 - 560 lux

EFFECT PARAMETERS MEASURED: The numbers of live and dead eggs were recorded daily with any dead eggs or dead larvae being discarded. Daily observation of larval mortality, behaviour (such as the "swim up" time) and appearance was made and any abnormal effects recorded.

VEHICLE CONTROL PERFORMED: yes
Reference substance (positive control):
no
Key result
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
40 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Survival, number hatched, length and weight
Key result
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
80 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Survival, number hatched, length and weight
Duration:
32 d
Dose descriptor:
other: MATC
Effect conc.:
>= 40 - <= 80 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Survival, number hatched, length and weight
Remarks on result:
other: Mean: 57 µg/L
Details on results:
- Mortality/survival at embryo and larval stages: The percent survival in the individual replicates ranged from 0 to 94% with an overall mean of 68%. The percent survival in the pooled replicates ranged from 0 to 81 % with an overall mean of 67%. 0% survival observed in 80 µg/L concentration.
- Days to hatch and numbers hatched: The percent hatchability in the individual replicates ranged from 62 to 100% with an overall mean of 85%. The percent hatchability in the pooled replicates ranged from 69 to 91% with an overall mean of 87%.
- Number of fish in swim-up stage at one or more time periods: The larvae had started to "swim up" on post hatch day 3 and most larvae were observed to be "swimming up" on post hatch day 6 which was considered to be "swim up day".
- Data for length and weight of surviving fish: No significant differences (P=0.05) in length and weight were detected in the nominal concentrations of 40 µg/L and below.
Reported statistics and error estimates:
The data (all replicates) for larval length and weight were tested for normality followed by Bartlett's test for homogeneity of variance. If the data had a normal distribution and the variance was homogeneous then the individual replicate data were analyzed using a parametric statistical procedure such as a t test following analysis of variance. If the data did not have a normal distribution or failed Bartlett's test for homogeneity of variance, then the individual replicate data were analyzed using a non-parametric procedure such as Wilcoxon's Rank Sum Test.
Validity criteria fulfilled:
yes
Conclusions:
32 d NOEC: 40 µg/L (Survival, number hatched, length and weight)
32 d LOEC: 80 µg/L (Survival, number hatched, length and weight)

Executive summary:

The toxicity of the test substance to the Fathead minnow (Pimephales promelas), was determined in a 32 day flowthrough fish early life stage toxicity test according to OECD guideline 210.

The study was conducted with six nominal concentrations of the test substance (5, 10, 20, 30, 40 and 80 μg/L), a dilution water control, and a solvent control at a temperature range of 24.0 - 25.3°C with 4 replicates. The mean measured concentrations were 4.8, 10, 19, 27, 36 and 73 μg/L respectively.

The percent survival in the individual replicates ranged from 0 to 94% with an overall mean of 68%. The percent survival in the pooled replicates ranged from 0 to 81 % with an overall mean of 67%. 0% survival observed in 80 µg/L concentration. The percent hatchability in the individual replicates ranged from 62 to 100% with an overall mean of 85%. The percent hatchability in the pooled replicates ranged from 69 to 91% with an overall mean of 87%. No significant differences (P=0.05) in length and weight were detected in the nominal concentrations of 40 µg/L and below.

The overall nominal No Observed Effect Concentration (NOEC) of the test substance was 40 µg/L and overall nominal Lowest Observed Effect Concentration (LOEC) was 80 µg/L based on Survival, number hatched, length and weight.

Description of key information

28-day NOEC (Oncorhynchus mykiss): 0.010 mg/L; OECD 204; Reliability = 1

28-day NOEC (Pimephales promelas): 0.040 mg/L; OECD 210; Reliability = 1

28-day NOEC (Cyprinodon variegatus): 0.021 mg/L; OECD 210; Reliability = 1

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
0.01 mg/L
EC10, LC10 or NOEC for marine water fish:
0.021 mg/L

Additional information

Chronic fish toxicity tests were conducted in two freshwater species and one marine species. The chronic NOEC value in Oncorhynchus mykiss in a 28d juvenile study was 0.010 mg a.s./L (based on nominal concentrations). Under the most recent data requirement, the freshwater species Pimephales promelas was tested in an ELS study with a NOEC = 0.040 mg a.s./L (nominal concentrations), based on development and growth parameters. The Cyprinodon variegatus showed similar toxicity to freshwater species, with a mean measured NOEC = 0.021 mg a.s./L. The chronic NOECs for fish are less than 0.1 mg a.s./L.