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Long-term toxicity to fish

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Reference
Endpoint:
fish life cycle toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16th March 1995 to 13th December 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPP 72-5 (Fish Life Cycle Toxicity)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The test solution in each aquarium was sampled a minimum of weekly until the lower level of the diluter system was activated to accommodate the F0 male fish. Subsequently, test solution samples were taken a minimum of weekly from one replicate aquaria of each treatment level and the controls from the corresponding upper and lower level. Samples were removed from alternating replicates weekly.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: diluter stock solution pf 6.4 mg/L prepared by dissolving approximately 0.7143 g test material with acetone to a total volume of 100 mL. 0.017 mL stock solution was added to the diluter's mixing chamber.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: fathead minnow
- Source: Springborn Laboratories, Inc.
- Pre-exposure reproductive performance

POST-HATCH FEEDING
- Type/source of feed: live brine shrimp nauplii (newly hatched fry); frozen brine shrimp and Zeigler® Brothers Prime flakes (juvenile adults)
- Frequency of feeding: approximately three times daily (newly-hatched fry); twice daily (juvenile adults). Frequency was reduced to twice daily and once daily, respectively, at weekends
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
272 d
Hardness:
33-35 mg/L CaCO3
Test temperature:
24-26 ºC
pH:
6.9-7.6
Dissolved oxygen:
7.5-7.9 mg/L
Nominal and measured concentrations:
0, 1.8, 3.5, 7, 14 and 28 µg/L
Details on test conditions:
TEST SYSTEM
- Embryo cups (if used, type/material, size, fill volume): 5 cm diameter glass jars with nylon screen bottoms
- Test vessel: 60 x 30 x 30 cm aquaria
- Material, size, headspace, fill volume: fill volume - 27 litres
- Type of flow-through: proportional diluter
- Renewal rate of test solution (frequency/flow rate): 7 volume replacements every 24 hours (pre-spawning phase); 6.2 volume replacements every 24 hours (spawning phase)
- No. of fertilized eggs/embryos per vessel: 50
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: well water
- Total organic carbon: 0.4-1.2 mg/L
- Metals: not detected
- Pesticides: not detected
- Alkalinity: 23-24 mg CaCO3/L
- Conductivity: 160-710 µmhos/cm

OTHER TEST CONDITIONS
- Photoperiod: 10.5-15.75 hours
- Light intensity: 50-100 footcandles

EFFECT PARAMETERS MEASURED:
- Percent hatching success was calculated based on the number of embryos introduced at test initiation when hatching was completed (day 5). If two or more embryos were unaccounted for in any egg cup, the percentage hatching success was calculated by dividing the number of live fry by the number of organisms accounted for.

RANGE-FINDING STUDY
- Test concentrations: 7.3, 11, 18, 37 and 51 µg/L
- Results used to determine the conditions for the definitive study: yes

POST-HATCH DETAILS
- No. of hatched eggs (alevins)/treatment released to the test chamber: four groups of 25 larvae per treatment level and controls.
- Release of alevins from incubation cups to test chamber on day no.: 5
Reference substance (positive control):
no
Key result
Duration:
272 d
Dose descriptor:
NOEC
Effect conc.:
5.7 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
larval development
Remarks:
growth of F1 larvae
Duration:
272 d
Dose descriptor:
LOEC
Effect conc.:
11 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
larval development
Remarks:
growth of F1 larvae
Duration:
272 d
Dose descriptor:
other: MATC
Effect conc.:
5.7 - 11 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
larval development
Remarks:
growth of F1 larvae
Details on results:
- Mortality/survival at embryo, larval, juvenile, and adult stages: at 30 days post-hatch, there was no effect on survival of F0 concentration up to and including 11 µg/L (88-92 % survival comparable to control). However, survival of the 27 µg/L at 30 days post-hatch was 26 % and significantly reduced; the same pattern was recorded at 59 days. By 272 days, survival in F0 adults was unaffected following exposure to concentrations up to and including 11 µg/L. At 27 µg/L, survival was reduced to 20 %. After 30 days post-hatch exposure of the F1 larvae, 100 % mortality was recorded at 27 µg/L.
- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: percent egg hatch in the F0 generation was not significantly affected at any exposure concentration (87-90 % in all test concentrations compared to the control). In the F1 generation, hatching success at 27 µg/L was 66 % a significant reduction when compared to the control (89 %).
- Observations on body length and weight of young and/or exposed parents at one or more time periods: at 30 days post-hatch, there was no effect on length of F0 concentration up to and including 11 µg/L. Statistical analysis of the 30 day larval length of 27 µg/L was not possible due to insufficient numbers of surviving fish; the same pattern was recorded at 59 days. In addition, there was no effect on weight of larvae at 59 days in concentrations up to and including 11 µg/L. By 272 days, length in F0 adults was unaffected following exposure to concentrations up to and including 11 µg/L. There was a statistically significant reduction in length and weight of 30 day larvae of the F1 generation following exposure at 11 µg/L with no treatment-related effects at 2.8 and 5.7 µg/L.
- Type of and number with morphological abnormalities: there were no treatment-related deformities.
- Detailed data on spawning, egg numbers, fertility, and fecundity: the number of eggs/spawn and spawns/female in the F0 parents was significantly reduced at 27 µg/L although the number of eggs/female was not affected at this concentration.
Validity criteria fulfilled:
not specified
Conclusions:
Under the conditions of the test, no effect on hatching success or survival of larvae at 30 days post-hatch was seen in F0 and F1 generations exposed to concentrations up to and including 11 µg/L. There was also no effect on length of F0 30 day larvae at 11 µg/L. Moreover, concentrations up to 11 µg/L had no effect on survival or growth of F1 adults at 59 and 272 days or on eggs/spawn and spawns/female. A concentration of 27 µg/L significantly reduced egg hatching, survival, eggs/spawn, spawn/female and growth at all time points. In addition, a concentration of 11 µg/L significantly reduced length and weight of F1 larvae at 30 days post-hatch. Therefore, based on the most sensitive endpoint of growth of the F1 larvae, the NOEC, LOEC and MATC value in this study were estimated to be 5.7, 11 and 5.7-11 µg/L, respectively.
Executive summary:

Fathead minnow were continuously exposed to five concentrations of Omite®, a dilution water control and a solvent control for a complete life-cycle (272 days). In addition, their F1 progeny were continued in exposure for 30 days post-hatch. The nominal concentrations of the test material for this study were 1.8, 3.5, 7.0, 14 and 28 µg/L (mean measured exposure concentrations were 1.4, 2.8, 5.7, 11 and 27 µg/L, respectively, which ranged from 76 to 95 % of the nominal concentrations).

Statistical comparisons to determine the significant toxicant effects were made using the pooled data from the dilution water and solvent controls for all endpoints except eggs per female and second generation (F1) hatching success. For these two endpoints, comparison was made to the solvent control. Dichotomous data were analysed using the appropriate contingency table test, and the continuous data were analysed using Williams' test.

Exposure at 27 µg/L significantly reduced F0 30 and 59 day post-hatch survival (26 %) as compared to the pooled control survival which was 90 and 91 % on days 30 and 59 post-hatch, respectively. After 59 days of exposure, there was no apparent effect on the length or weight of the exposed fish at any concentration tested below 27 µg/L compared to the controls. At test termination, F0 survival at 27 µg/L was 20 % compared to pooled control survival (92 %) and was significantly reduced. F0 growth at all test concentrations below 27 µg/L was comparable to the control. The number of eggs per spawn and spawns per female were also significantly reduced at 27 µg/L, however, no significant difference was established at any test concentration for eggs per female compared to the solvent control.

The number of live F1 fry at hatch was statistically determined to be reduced at 27 µg/L (66 %) compared to the solvent control (89 %) and following 30 days post-hatch exposure, F1 larval survival at this treatment level was reduced (0 %) compared to the pooled control (89 %). An effect on the growth (total length and wet weight) of F1 larval fish at 11 µg/L was statistically established based on comparison to the pooled control. Statistical analysis indicated no significant difference in the total length of larval fish exposed to test concentrations at or below 5.7 µg/L. However, a significant reduction in wet weight was indicated at 2.8 and 5.7 µg/L. The weight difference at 2.8 µg/L was not considered toxicant related as the response did not follow the concentration gradient. At 5.7 µg/L, the mean wet weight was 0.23 g compared to 0.24 g with the pooled control (4 % reduction). This difference was considered biologically insignificant as it is highly unlikely that a 0.1 g reduction in weight would have any impact on the fathead minnow population.

The most sensitive indicator of toxicity was the reduced F1 generation growth (total length and wet weight). Based on these data, the MATC was estimated to be >5.7 µg/L (NOEC) and <11 µg/L (LOEC). The geometric mean MATC was 7.9 µg/L.

Description of key information

NOEC 5.7 µg/L (sublethal effects); study conducted in accordance with EPA OPP 72-5; Dionne, 1996

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
5.7 µg/L

Additional information

The chronic toxicity of propargite to rainbow trout was determined in a standard 21 day chronic toxicity study. The life-cycle toxicity of propargite to fathead minnow was determined in a 272 day study. Both studies were performed to standardised guidelines and GLP and were both assigned a reliability score of 1. The results were comparable between the two studies and so the life-cycle fathead minnow study was selected as the key study on the basis that it was performed over a longer time period. In addition, the early life-stage toxicity of propargite to fathead minnow was determined in a GLP study performed to ASTM guidelines; this study was assigned a reliability score of 2 and so was considered to be a supporting study.

In the key study, fathead minnow were continuously exposed to five measured concentrations (mean measured concentrations of 1.4, 2.8, 5.7, 11 and 27 µg/L) of test material for 272 days. In addition, their F1 progeny were continued in exposure for 30 days post-hatch. Exposure at 27 µg/L significantly reduced F0 30 and 59 day post-hatch survival (26 %) compared to the controls. After 59 days of exposure, there was no apparent effect on the length or weight of the exposed fish at any concentrations tested below 27 µg/L compared to the controls. At test termination, F0 survival at 27 µg/L was 20 % compared to pooled control survival (92 %) and was significantly reduced. F0 growth at all test concentrations below 27 µg/L was comparable to the control. The number of eggs per spawn and spawns per female were also significantly reduced at 27 µg/L, however, no significant difference was established at any test concentration for eggs per female compared to the solvent control. The number of live F1 fry at hatch was statistically determined to be reduced at 27 µg/L (66 %) compared to the solvent control (89 %) and following 30 days post-hatch exposure, F1 larval survival at this treatment level was reduced (0 %) compared to the pooled control (89 %). An effect on the growth (total length and wet weight) of F 1larval fish at 11 µg/L was statistically established based on comparison to the pooled control. There was no significant difference in the total length of larval fish exposed to test concentrations at or below 5.7 µg/L. However, a significant reduction in wet weight was indicated at 2.8 and 5.7 µg/L; the weight difference at 2.8 µg/L was not considered toxicant related as the response did not follow the concentration gradient. At 5.7 µg/L, the mean wet weight was 0.23 g compared to 0.24 g with the pooled control (4 % reduction); this difference was considered biologically insignificant as it is highly unlikely that a 0.1 g reduction in weight would have any impact on the fathead minnow population. The most sensitive indicator of toxicity was the reduced F1 generation growth (total length and wet weight). Based on these data, the MATC was estimated to be >5.7 µg/L (NOEC) and <11 µg/L (LOEC). The geometric mean MATC was 7.9 µg/L.

In the supporting 21 day rainbow trout study, twenty organisms were exposed to five measured concentrations (14, 21, 32, 52 and 100 µg/L) of test material using an intermittent-flow proportional diluter apparatus. Biological observations were recorded at test initiation and every 24 hours thereafter. After 96 hours, 100 % mortality was observed at the highest test concentration. By test termination, 100 % mortality was also observed at 32 and 52 µg/L with 20 % mortality at 21 µg/L. No mortality was observed at 14 µg/L but sublethal effects were observed at this treatment level. The LC50 was determined to be 21 µg/L and the NOEC to be <14 µg/L.

In the supporting early-life cycle fathead minnow toxicity study, a two litre proportional diluter system was used to maintain constant mean measured test concentrations of 1.9, 3.2, 7.5, 16 and 28 µg/L. Hatchability of fathead minnow eggs was not significantly affected by exposure to the test material. Survival of fry between hatch and day 35 was significantly affected at 28 µg/L when compared to controls. Length and weight data were not available for statistical analysis for the 28 µg/L test concentration since no fish remained at the termination of the study. All remaining test concentrations did not show a significant decrease in length and weight of the fish after 35 days exposure. Based on these data, the MATC values are 16 and 28 µg/L of test material.

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