1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish life cycle toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Jul 1987 to 28 Oct 1987

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)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test aquaria at test initiation, weekly for the next five weeks, and monthly thereafter. On the other weeks when the complete set of samples was not collected, samples were taken from one replicate aquarium of each test concentration and control, alternating replicates weekly. During the spawning period, this overall sampling pattern included the aquaria in both tiers (upper and lower) of the test system.

Vehicle:

no

Details on test solutions:

STOCK SOLUTION PREPARATION
Stock solutions were prepared by placing 100 mg of the test substance per liter of seawater in a 200 liter Nalgene tank and mixing with four magnetic drive pumps. After 48 hours of mixing, the test substance concentration of the saturated solution was analyzed and diluted to 25 mg/L.

DILUTER SYSTEM
The stock solution was introduced to the dilution system by utilizing a pre-dilution chamber into which the toxicant stock solution was pumped by a peristaltic pump. A calibrated volume of the stock solution was delivered with each diluter cycle to a glass mixing chamber. It was then diluted to equal the highest test concentration which in turn was diluted proportionally to produce the test concentration range. Flow splitting chambers were used to promote further mixing and to equally distribute the test solutions through glass delivery tubes equally distribute the test solutions through glass delivery tubes.

Test organisms (species):

Cyprinodon variegatus

Details on test organisms:

TEST ORGANISM
- Common name: Sheepshead minnow
- Source: A brood group of sexually developed adult was purhased. This brood stock was subsequently used for spawning to obtain embryos to initiate the chronic exposure.
- Age of eggs when experiment started: ≤ 24 hours
- Source of eggs: The eggs were obtained from the sheepshead minnow brood stock which had been divided into 14 groups of 7 females and 2 males each.

ACCLIMATION
- Acclimation period: 14 days
- Salinity: 31 - 34‰
- Temperature: 27 - 30°C
- pH: 7.3 - 7.9
- Dissolved oxygen: 64 - 70% of saturation

FEEDING DURING TEST:
- Food type (F0 and F1): Larvae were fed live brine shrimp nauplii for 28 days post-hatch
- Food type (juvenile and adult fish): Commercial flakes
- Frequency (all test organisms are the same): Three times daily (twice on weekends)

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

100 d

Remarks on exposure duration:

95 days F0 exposure, 91 days post-hatch F0 exposure, five additional days to complete the F1 exposure

Test temperature:

- Upper level: 26 - 30°C
- Lower level: 26 - 31°C

pH:

7.9 - 8.1

Dissolved oxygen:

- 4.2 - 7.1 mg O2/L
- Mean concentration 97 - 98% of saturation (Oxygen saturation at 30°C and 30 ppt salinity is 6.2 mg/L. The lowest observed oxygen value (4.2 mg/L) represents 68% of saturation. The highest oxygen concentration (7.1 mg/L) is 115% of saturation.

Salinity:

30 - 32‰

Nominal and measured concentrations:

- Nominal concentration: 0.019, 0.038, 0.075, 0.15, 0.30 and 0.60 mg/L
- Measured concentration: 0.016, 0.038, 0.068, 0.15, 0.29 and 0.55 mg/L, respectively (See Table 1 in any other information on materials and methods incl. tables)

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquarium
- Size of vessel: 60 x 30 x 30 cm
- Material and fill volume of vessel: Constructed from glass with silicone sealant seams and were equipped with a 15-cm high end-drain to maintain the test solution volume at 27 L
- Exposure system: The exposure system was a two-tiered system, consisting of an upper and a lower level water bath. A total of 28 aquaria in the two tiers combined. The positions of the 14 aquaria in each water bath were assigned by a random selection of number and letter combinations relating to test concentration level and replicate
- Water bath: 31°C maintained by means of a mercury column thermoregulator and two 1000-watt quartz heaters
- Upper level functions: Embryo incubation cups were used to expose the fertilized eggs. These cups consisted of round glass jars, 5 cm in diameter and with a height of 8 cm, equipped with stainless steel wire handles and 40 mesh nylon screen bottoms. A 16 x 7.5 x 7.5 cm embryo incubation chamber was attached at the head of each upper level aquarium originally designed to hold two of these embryo incubation cups. Instead, the embryo incubation cups were suspended from the embryo incubation chambers with stainless steel wire hangers into the main area of the exposure aquaria where they were aerated from below to circulate water around the embryos. This modification was implemented when it became evident that the water temperature in the incubation chambers could not be maintained at the test temperature range of 28 - 32°C due to the position of the chambers above the surface of the aquaria and the temperature regulation provided by the heated water bath. Each aquarium in the upper level of the system was assigned two larval chambers for maintaining < 28 days old F0 and F1 larvae. These chambers measured 30 x 14.5 x 17 cm and were constructed of glass using silicone sealant seams and 40-mesh nylon screening for the ends. During the F1 larval exposure, an 8.5-mesh nylon screen divider was used to divide the tank into two sections; one for the two larval chambers and one for adult fish in the pre-spawn or post-spawn phase.
- Lower level functions: The lower section of the test system was activated during the spawning phase of the exposure. Fourteen aquaria provided the space for 28 spawning "baskets" made of 8.5 mesh nylon screening. Each basket, measuring 30 cm square with a water depth of ~12 cm, was placed over a removable egg collection tray. These trays were constructed with 3 cm high glass sides and 40-mesh nylon screening bottoms and were designed to secure the eggs produced by the parental fish in the spawning baskets above, and to provide a means of easily consolidating and collecting spawns.
- Type of flow-through: Proportional diluter
- Dilution factor: 0.5
- Flow rate (When only the upper system was in use): On average 194 diluter cycles daily provided 7.2 tank volume turnovers per 24 hours or 90% replacement in 8 hours
- Flow rate (when both upper and lower levels of system were in use): On average 278 diluter cycles daily provided 5.1 tank volume turnovers per 24 hours or 90% replacement in 9 hours
- No. of organisms per vessel: 50 embryos
- No. of vessels per concentration: 2
- No. of vessels per control: 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water used during this study was natural filtered seawater. The seawater was collected from the Cape Cod Canal, Bourne, Massachusetts. Prior to use, the seawater was filtered through a series of polypropylene core filters (20 and 5 µm) and then recirculated within an epoxy lined concrete reservoir. The filtered seawater was pumped to the test system through PVC piping, an activated carbon filter, and a polypropylene heat exchanger system.
- Quality of the filution water: The quality of the dilution water for use in toxicity tests with marine organisms has been established at the test facility by the successful reproductive, survival and growth of representative species, e.g., mysid shrimp and sheepshead minnow. Routine chemical analyses of the seawater for potential toxic substances (metals, pesticides and PCB's) are conducted on representative samples of the seawater. None of these analyses have detected measurable quantities of these toxicants in any water sample analyzed. Total organic carbon was measured in filtered seawater at the test facility once during this study period.
- Intervals of water quality measurement: At test initiation, all water quality parameters were determined in each test aquarium. During the exposure, temperature and dissolved oxygen were measured daily, and salinity and pH weekly, in alternating replicate tanks of treatments and control.
- Test monotroing: The test system was continuously monitored for test aquaria temperature fluctuations outside of the acceptable 26 - 32°C range. An alarm system consisted of thermostat controls set for the high and low of the temperature range using temperature probes which were placed in two upper and lower level test aquaria. If a temperature fell below 26°C or rose above 32°C , then the thermostat controls triggered a sound alarm within the building and an electronic signal to a communication center which notified key personnel.

OTHER TEST CONDITIONS
- Photoperiod: 12 hours daylight and 12 hours darkness
- Light intensity: 34 to 60 footcandles (upper level); 100 to 200 footcandles (lower level)
- The entire test system was enclosed in black polyethylene curtains to prevent disturbance of the fish and to minimize the interference of laboratory lighting with the intended photoperiod.

TEST PROCEDURE AND EFFECT PARAMETERS MEASURED:
- Test initiation: The chronic study was initiated by incubating 100 eggs, equally divided between two egg cups, in each aquarium (1,400 eggs in total). The eggs were ≤ 24 hours old and were obtained from the sheepshead minnow brood stock which had been divided into 14 groups of 7 females and 2 males each. Eggs from the 14 groups were pooled and a representative subsample of 100 eggs was microscopically examined to estimate the percentage of successfully fertilized eggs (embryos). This examination indicated that 73% of the eggs were fertilized. Twenty-eight embryo incubation cups, with treatment and replicate designations, were randomly arranged in a tray containing 30°C seawater. Embryos were distributed to the cups, five at a time, until each cup contained 50 embryos. Each day until hatching was completed (4 days), the embryos in each cup were counted and dead embryos were removed. Percentage hatching success was calculated for each duplicate aquarium (number of live fry per number of eggs incubated x 100). Twenty-five of the newly-hatched fry in each incubation cup group were impartially selected and placed in their respective growth chambers.

- Day 32 - 52 exposure: After 28 days of post-hatch exposure, the juvenile fish in the two growth chambers within each replicate tank were combined. From each group, 25 fish were randomly selected to be continued in exposure by using a random number generating technique. Each of these groups was photographed, using the photographic method of McKim and Benoit (1971), in order to measure the total length of individual fish and to accurately count the fish for determining percentage survival. The fish discontinued from exposure were euthanized with Tricaine Methanesulfonate and were manually measured with a millimeter ruler and weighed on a balance. These groups of terminated fish were pooled by treatment level and frozen for possible residue analysis. On test day 46 post-hatch, territorial behavior was being exhibited by several males at two treatment levels of the study. A second set of photographs for growth and survival determinations of pre-spawning fish, originally scheduled for day 56 post-hatch was taken on day 48 post-hatch.

- F0 spawning: On day 49 post-hatch, the first spawning group (2 males and 5 females) from each test aquarium was placed in one section of the corresponding spawning chamber in the lower level of the test system, where they remained for 14 days. The system provided sufficient space for two separate spawning groups per replicate aquarium, and the second groups were established on day 52 post-hatch (i.e., day 56 of exposure). During this life cycle study, a total of four spawning groups per replicate aquarium, or eight groups per treatment, were established and spawning recorded. Spawns were removed and counted daily. Eggs not used for F1 exposure were pooled by treatment and frozen for possible future residue analysis. Females killed during spawning were not replaced in the spawning group, however, males were replaced in order to maximize egg fertilization success. Female mortality was always the direct result of male aggression. Reproductive success was calculated based on the number of eggs per number of females alive per spawning day. For example, if on any given day during the 14-day spawning period a total of 78 eggs were counted in the spawning tray, and four females were alive at that day, then the reproductive success for that day would be 78/4, or 19.5. The mean reproductive success for each spawning group represents the mean of 14 consecutive reproductive success ratios. Exposure of the first generation (F0) fish was terminated on test day 95 (day 91 post-hatch) after spawning had been assessed. Each fish was individually measured for total length, wet weighed (blotted dry) and internally examined to verify sex and gonadal condition. These fish were pooled by treatment level and frozen for possible residue analysis.

- F1 hatching success: In addition to determination of hatching success of embryo groups used to perform the F1 early life-stage exposure, hatching success was determined frequently throughout the 14-day spawning periods in groups of 50 eggs except at the treatment levels where spawning was reduced due to the toxicant. Smaller groups of eggs were incubated if less than 50 eggs were available. Moreover, several groups of control embryos were transferred and incubated at the highest two treatment levels to corroborate the response of F0 embryos exposed at these concentrations. Hatching success was determined as the ratio of number of live fry at the end of the 4-day hatching period to the number of egges exposed initially, and was calculated separately for each group of eggs exposed.

- F1 early life stage exposure: Exposure of the second generation (F1) fish began by incubating groups of 50 embryos on the day they were spawned. Two embryo groups of 50 eggs were incubated concurrently per replicate concentration or control. These embryo groups were obtained from two different spawns. Following hatching of the embryos, F1 exposure was continued by placing the two groups of newly hatched larvae into corresponding larval growth chambers. After twenty-eight days of post-hatch exposure, each group was euthanized with Tricaine Methanesulfonate and the fish were individually measured with a millimeter ruler and weighed on a balance. Each group was frozen for possible residue analysis.

-Evaluation of short-term exposure: An investigation, supplemental to the full life cycle study, was conducted to evaluate the effect of short-term (i.e., 18 days) exposure of sheepshead minnow to the test substance. Two groups of ≤ 24 hours old control F1 embryos (25 per group) were transferred and incubated at mean measured concentrations of 0.55 and 0.29 mg/L the test substance in the chronic exposure system. After hatching, fry were exposed at these concentration levels for 14 days before being transferred to larval chambers in a recirculating seawater system containing no toxicant. Total exposure time of embryos and larvae was 18 days. Thirty-two fish temporarily exposed to 0.55 mg/L and thirty-four fish temporarily exposed to 0.29 mg/Lthe test substance were reared to maturity in this system. Following maturation, two
spawning groups of two males and five females were established for each group of exposed fish. Reproduction was evaluated for 14 day
spawning periods for each group of spawners in the manner described above. Similarly, hatching success was evaluated in several embryo groups during the 14-day spawning periods.

RANGE - FINDING STUDY
- Test description: Acute 96-h toxicity tests under static and flow-through conditions were performed to establishe a test concentration range fo the chronic exposure. A solvent (acetone) was used to assist in the solubilization of the test material, and a solvent control was maintained in addition to a seawater control.
- Results: The results of the flow-through exposure indicated no statistically significant (P ≤ 0.05) difference in embryo hatachability and larval survival and growth between sheepshead minnow exposed to mean measured test concentrations ≤ 0.60 mg/L and the seawater control. However, lengths of solvent control fish were 27 mm (SD = 2), which was significantly longer than those of the seawater control fish, measuring 24 mm (SD = 2). Solvent control fish were also longer than the fish at all treatment levels (23 to 24 mm). Based on these data, the nominal concentration range of 0.60, 0.30, 0.15, 0.075, 0.038 and 0.019 mg/L was selected for the chronic exposure. In addition, at test termination solvent control fish were significantly larger than the seawater control fish and the fish at all treatment levels, indicating an apparent solvent effect on fish growth, Therefore, a method was developed to prepare toxicant stock solutions of the test substance in seawater without the use of a solvent.

Reference substance (positive control):

no

Key result

Duration:

100 d

Dose descriptor:

NOEC

Effect conc.:

0.068 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

An overwiew of the results is provided in Table 2 - Table 4 in "Any other information on results incl. tables"

BIOLOGICAL OBSERVATIONS
In this study, hatching was found to be completed within four days of egg release. The fry and juvenile fish were monitored daily and a
record of the lengths of fish alive at day 28 post-hatch was obtained by photographic means for any fish used for the full life cycle study. Fish which were removed from exposure during thinning at day 28 post-hatch were measured and weighed also. On day 48 post-hatch, after fish had reached sexual maturity, lengths of all surviving fish was obtained again photographically, and an accurate percentage survival was again established. Reproductive success data of four spawning groups per replicate concentration or control was obtained in the period following day 48 post-hatch until test termination. The viability of a representative number of the spawns produced was established also. Moreover, four groups of 25 larvae were maintained in each control and test solution for a period of 28 days post-hatch, and their survival and growth was determined. At test termination all surviving fish were measured and weighed.

F0 EXPOSURE
Exposure to all concentrations of the test substance tested (0.016 - 0.55 mg/L) did not adversely affect embryo hatchability or survival of fish at any stage. Fish survival after thinning of the population was calculated based on the number present on day 1 post-hatch, after adjusting the number of surviving fish as shown in the following example: Control. Replicate A: On day 1 post-hatch, 50 fish were alive. Only 49 fish were alive on day 32 (98%). After thinning, 25 fish remained. On day 52, all 25 fish were still alive. The "adjusted survival" was calculated by multiplying the number of fish alive by the thinning ratio: (49/25) x 25 = 49, i.e., 98%. Similarly, day 95 survival was 23 fish. Adjusted survival on that day was therefore (49/25) x 23 = 45 (90%).
By exposure day 52 (48 days post-hatch), nearly all exposed fish in all treatment levels and the controls completed their sexual development, as evidenced by clear sexual dimorphism and aggressive behavior of the male fish. The concentrations of the test substance tested did not appear to significantly alter the time required for the fish to reach this developmental stage. However, during spawning trials, females were occasionally lost due to persistent male territorial behavior (chasing, biting). Female mortalities could clearly be ascribed to this behavioral pattern, rather than to any toxicant-mediated stress, as evidenced by the random occurrence of such mortalities in both concentration and control spawn groups.
Lengths and weights of F0 sheepshead minnow were unaffected by any of the exposure concentrations considered. Statistical comparisons of growth data confirmed no toxicant-related significant reduction in either fish lengths or weights for any interval and at any of the test concentrations tested. Fish lengths ranged from 24 to 26 mm at day 28 post-hatch, and from 34 to 35 mm at day 48 post-hatch. After 91 days post-hatch exposure, male fish ranged between 38 and 41 mm, while females were 34 to 39 mm in size. At test termination, mean weights of male sheepshead minnow exposed to the highest test concentration and control fish were 1.15 g and 1.11 g, respectively. Noteworthy is the discrepancy in female weights between control fish and those exposed to the highest test concentration of 0.55 mg/L the test substance. Exposed female fish were 59% heavier than control fish. This can be attributed to the large number of unspawned eggs present in the females at this test concentration which significantly reduced spawning. Throughout the study period, no abnormal appearance or behavior was observed in any of the treatment levels.
Reproductive success (i.e., spawning success) was determined for each spawning group from the number of eggs produced per day by that spawning group, dividing this number by the number of females alive on the day of observation (i.e eggs per female), and calculating the mean number of eggs produced per female throughout the 14 day spawning period. A total of eight spawn groups were evaluated (four per replicate aquarium). Spawn group 2 was initiated within a few days of spawn group 1, using females which were in spawning condition. Spawn group 3 was started several weeks later, while spawn group 4 used females which previously had been placed in a spawn group. This practice was considered acceptable as sheepshead minnow spawn continuously. In contrast to the absence of survival and growth effects, exposure to the highest three test concentrations (0.55, 0.29, and 0.15 mg/L) significantly reduced the reproductive success (mean embryos produced/female/day) of sheepshead minnow. Statistical evaluation of the spawn data of the four spawn groups by two-way ANOVA indicated no statistical differences between spawn groups (P ≤ 0.05), and the data were subsequently pooled for statistical comparison of treatment levels and controls. As indicated above, 0.15 mg/L the test substance significantly affected spawning success of the sheepshead minnow, and the Maximum Acceptable Toxicant Concentration (MATC) of the test substance is therefore estimated to be > 0.068 mg/L and < 0.15 mg/L, with a geometric mean MATC of 0.10 mg/L.
A large number of eggs (up to 1,847) produced by F0 females were maintained in the test solutions to evaluate their viability (i.e., hatching success). The viability of F1 embryos produced by these fish was significantly reduced by exposure to 0.55 and 0.29 mg/L the test substance. Hatching success data were statistically analyzed in the same manner as the spawning data. The reduction in numbers of percent eggs hatched at the highest two exposure concentrations was 61 and 65%. Groups of 50 embryos spawned in control water were transferred within 24 hours to 0.29 and 0.55 mg/L the test substance to confirm earlier observations (for F0 fish) that embryos produced by unexposed females were viable. The hatching success of these embryos was 80% and 76%, respectively, and was indeed very similar to the hatching success of F0 embryos exposed at these test concentrations. Based on F1 hatching success, the MATC of the test substance is estimated to be > 0.15 mg/L and < 0.29 mg/L (geometric mean MATC = 0.21 mg/L).
While the effect concentration for hatching success is higher than that for spawning success, the two effects would be expected to accentuate one another. For example, if the reduction in the number of embryos produced at a given concentration was 20% (compared to the control) and only 50% of embryos produced were viable, than the overall reduction in fish population would be 60%. Because of the synergism between spawning success and embryo hatchability, the reduction in spawn production at 0.29 and 0.55 mg/L should be considered substantially more significant with regards to their impact on population dynamics for that fish species than would the next lower concentration of 0.15 mg/L.

TEMPORAL EXPOSURE OF EMBRYOS AND LARVAE
Exposure of embryos and larvae to the two highest test concentrations for a period of 14 days post-hatch (18 days in total), followed by maintenance in clean dilution seawater until fish had reached maturity, did not negatively affect their ability to reproduce successfully, nor did it negatively affect the hatching success of their progeny. Female sheepshead minnow which were exposed for a short duration produced as many eggs as did unexposed fish, while the hatching success of these eggs was very similar to the hatching success of the controls. Apparently, any adverse effects caused by the test material did not occur during the earliest, generally most sensitive life stages.

F1 EXPOSURE
Survival and growth of fish was evaluated for 28 days post-hatch. When possible, four groups of 25 fish were so evaluated. Reduced spawning at the highest two exposure concentrations prevented performance of an early life-stage exposure at these concentrations. No statistically significant adverse effects were observed among second generation larval fish.
Survival was 100% at 0.15 mg/L the test substance vs. 95% in the controls. Lengths and weights of fish exposed to that concentration did not differ from those of control fish.

Reported statistics and error estimates:

See Statistical analysis in "Any other information on materials and methods incl. tables".

Table 2. Mean percentage survival of F0 generation of sheepshead minnow based on the measured concentrations

Measured concentration (mg/L)	Viability of embryo	Viability of larvae, at day 28	Viability of larvae, at day 48^a	Viability of larvae, at day 91^a
Control	75	97	97	83
0.016	80	95	95	71
0.038	74	94	94	73
0.068	78	95	93	74
0.15	74	91	91	54
0.29	80	98	98	65
0.55	74	96	96	81

a.  Based on normalized data to adjust for thinning of the population.

Table 3. Result of mean lengths and weights of F0 sheepshead minnow

Measured concentration (mg/L)	Length (mm) Mean (S.D.)				Weight (g) Mean (S.D.)
	Day 28	Day 48	Day 91		Dy 28	Day 91
			Males	Females		Males	Females
Control	25 (1)	34 (1)	39 (1)	34 (0)	0.22 (0.00)	1.11 (0.05)	0.73 (0.01)
0.016	25 (0)	34 (1)	38/ (0)	35 (1)	0.23 (0.00)	1.08 (0.06)	0.74 (0.01)
0.038	26 (1)	35 (0)	40 (2)	36 (1)	0.26 (0.04)	1.26 (0.10)	0.76 (0.05)
0.068	25 (1)	35 (1)	39 (1)	35 (1)	0.21 (0.01)	1.09 (0.09)	0.72 (0.05)
0.15	25 (1)	35 (0)	41 (2)	35 (1)	0.24 (0.03)	1.37 (0.32)	0.81 (0.080
0.29	25 (1)	35 (0)	40 (1)	34 (4)	0.23 (0.00)	1.24 (0.03)	0.73 (0.09)
0.55	24 (0)	34 (0)	39 (3)	39 (2)	0.22 (0.00)	1.11 (0.20)	1.16 (0.13)

Table 4.Reproductive success of F0-generation and survival of F1-generation of sheepshead minnow

Concent. mg/L	Eggs produced total^1	Eggs/ female/ day^2	Eggs incubated total^3	Eggs Hatched (%)^4	F1-survi- val, 28- day post-hatch (%)
Control	7740	13.9	1847	75	95
0.016	5262	11.3	1593	77	98
0.038	6393	11.8	1728	75	93
0.068	6074	13.2	1400	79	92
0.15	4515	8.9	1045	80	100
0.29	2435	5.1	760	26	100
0.55	620	1.2	338	29	0

1 Eggs produced based on 8 spawnings of 14 days each
2 Based on 8 14-day spawning and corrected fot the no. of females alive on each day of spawning
3 Eggs incubated were obtained during 8 14-day spawnings
4 Percent eggs hatched are based on no. of live fry following 4 days of incubation and the initial no. of embryos exposed

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 100-d NOEC of the test substance was determinded to be 0.068 mg/L, and the LOEC was determined to be 0.15 mg/L

Executive summary:

The effects of the test substance on hatching success, survival, growth and reproductive success of first generation (F0) of sheepshead minnow (Cyprinodon variegatus) and the hatching success, survival and growth of their progeny (F1) was studied for 100 days (95 days F0 exposure, 91 days post-hatch F0 exposure, five additional days to complete the F1 exposure). The study was performed according to EPA guidelines and in compliance with GLP. Before the exposure the fish were acclimatized to natural seawater for 14 days. The conditions of the test were as following: salinity 30 - 32‰, temperature of 27 - 31°C, pH 7.9 - 8.2 and dissolved oxygen 64 - 70 % of saturation for 14 days. The test concentrations of the test substance were 0.016, 0.038, 0.068, 0.15, 0.29 and 0.55 mg/L based on measured concentrations and the number of animals were as following: days 0 - 5 F0 200 eggs/dose level; days 5 - 32 F0 100 larvae/dose level; days 32 - 52 F0 50 juvenile/dose level; days 52 - 85 F0 50 adults/dose level; days 55 - 100 F1 100/dose level. F0 and F1 larvae were fed live brine shrimp nauplii three times daily for 28 post hatch. Juvenile and adult fish were fed flakes withthe same schedule. All water quality parameters were determined before the test. Temperature and dissolved oxygen were measured daily during the study, salinity and pH weekly. Mortality and general symptoms of toxicity were evaluated daily. At days 28 and 48 post-hatch, the lengths of the fish alive were obtained photographically. Fish which were removed from exposure during thinning at day 28 post-hatch were measured and weighed also. Reproductive success data of 4 spawning groups per replicate concentration or control was obtained in the period following day 48 post-hatch until test termination. The concentration of the test substance in the test medium was determined once a week in all the replicates.

There was not any concentration dependent effect in the embryo hatchability or survival of fish. Additionally nearly all the fish completed their sexual development after 52 days of exposure. There was no statistically significant reduction in the length and weight of the larvae exposed different concentrations compared to the control. Reproduction was affected at concentration levels of 0.15 to 0.55 mg/L and the viability of F1 embryos was significantly reduced at 0.29 and 0.55 mg/L respectively. There was no difference in survival and growth rates of F1 juveniles at 0.016 - 0.15 mg/L and the control group.

Based on the findings, reproduction is the most sensitive endpoint, and NOEC value of the test substance was determined to be 0.068 mg/L.

Reference 2

Endpoint:

fish life cycle toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 March 2013 to 24 January 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 229: Fish Short Term Reproduction Assay

Qualifier:

according to guideline

Guideline:

other: OECD Draft Proposal for Fish Two-Generation Test

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 890.1350 – Fish Short-Term Reproduction

Qualifier:

according to guideline

Guideline:

other: EAP OPPTS 890.1500 – Male Pubertal (Rat)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Throughout the study, all samples were collected from mid-depth at each interval, placed in glass scintillation vials, and either processed immediately for analysis or stored refrigerated as reserve samples. Back-up samples were collected at each sampling interval starting on day 116 of the study. Additional water samples were collected as needed during the test to confirm concentrations when previous results were questionable. Samples of the stock solution were collected for analysis prior to and during the exposure period to confirm the concentrations being delivered to the diluter system. Samples were placed in glass vials and processed immediately for analysis. Additional stock samples were also collected during the study for confirmation of stock concentration.

F0 GENERATION
Water samples were collected from one replicate test vessel in each treatment and control group prior to the start of the exposure period to confirm the concentrations after conditioning the diluter system. Water samples also were collected from each replicate test vessel at the beginning and end of the exposure period (Days 0 and 21), and from two alternating replicate test vessels in each treatment and control group on Days 7 and 14 to measure concentrations of the test substance. The F0 generation was maintained until hatch of the F1 generation embryos was confirmed (Day 26). Additional water samples were collected on Day 26, and during the recovery phase. A set of back up samples was also collected on Day 26 and stored for possible analysis. Water samples were also collected from each replicate during the recovery phase.

F1 AND F2 GENERATIONS
Water samples were collected from one replicate test vessel in each treatment and control group prior to the start of the exposure period to confirm the concentrations after conditioning the diluter system. Water samples also were collected from one replicate test vessel in each treatment and control group at the beginning the exposure period, and then approximately weekly to measure concentrations of the test substance. Sample collection typically rotated among the replicates in each group at each sampling interval. Additional samples were collected as needed to confirm concentrations when a delivery or sampling error occurred, or to confirm previous results. All samples were collected at mid-depth from the test vessels and were either processed immediately for analysis, or held under refrigeration until analyzed.

Vehicle:

no

Details on test solutions:

Stock solutions of the test substance in dilution water were prepared ninety-seven times during the study at appropriate concentrations to deliver the nominal test concentrations. The test substance was heated to 30°C and weighed into a tared beaker. The calculated amount of test substance was thoroughly rinsed into 160-L of dilution water. The stock solutions were mixed overnight using a top-down mixer.
On Day 63 of the study mixing procedures were modified due to slightly reduced recoveries noted in expired stock solutions. The calculated amount of test material was thoroughly rinsed into a shear pump containing 30-L of dilution water. The stock solution was allowed to mix by the forced vortex of the shear pump for approximately 5 hours. After mixing, the solution was drained from the shear pump into a 175-L stainless steel drum and brought to volume with dilution water.
The stock solutions appeared clear and colorless. The stock solutions were held under ambient conditions, were protected from light, and were mixed continuously using a topdown electric mixer. The stock was pumped into the diluter mixing chambers assigned to the treatment groups at appropriate target rates, and was mixed with well water in the mixing chambers, to achieve the nominal test concentrations of 9, 26, 78, 233 and 700 µg/L, respectively.

F0 GENERATION
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 vessels. The flow of dilution water into each mixing chamber was controlled using rotameters, which were calibrated prior to the test and approximately weekly during the F0 generation. After mixing, the test solution in each mixing chamber was pumped into the appropriate replicate test chamber using a peristaltic pump.
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 solution that was pumped into each replicate test vessel 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 flow rate for the replicates. Delivery of test solutions to the test vessels was initiated at least 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.

F1 AND F2 GENERATIONS
The stock solution was diluted with well water in the mixing chambers with dilution water prior to delivery to the test vessels. The flow of dilution water into each mixing chamber was controlled using rotameters, which were calibrated prior to the test and approximately weekly during the test. On approximately day 176 of the study (day 158 of the F1 generation) calibrations of the test system were performed daily to improve analytical recoveries. The flow of dilution water into each mixing chamber was controlled using rotameters. Delivery of test solutions to the test vessels for the F1 and F2 generations was initiated at least five days prior to the introduction of the test organisms in order to achieve equilibrium of the test substance in the test system.
After mixing, the flow from each mixing chamber was split to deliver test water to the replicate test vessels. 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 flow rates. The general operation of the delivery system was checked visually at least two times per day during exposure of each generation and at least once at the beginning and end of exposure of each generation. Periodically during each generation of the test, the organisms were transferred to clean test vessels to prevent the build-up of bacterial/fungal growth.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Source: Laboratory-acclimated fish originally obtained from a supplier in Osage Beach, Missouri
- Age at study initiation: Approximately 6 months
- Mortality and Health: < 1% mortality during the 7 days prior to pre-exposure, and the fish showed no signs of disease or stress. The fish did not receive treatment for disease during the acclimation period, the two-week pre-exposure period or the exposure period

ACCLIMATION
- Acclimation period: Approximately 8-week
- Acclimation conditions: The test fish were held in water from the same source and at approximately the same temperature as used during the test
- Lighting: Under ambient laboratory lighting (16 horus/ 8 hours, light/dark, and a 30-minute transition period of low-light intensity)
- Temperature: 24.6 - 25.4°C
- pH: 8.3 - 8.7
- Dissolved oxygen concentrations: ≥7.5 mg/L (≥92% of air saturation)
- Type of food: Commercial flake food (Sera Vipan) and brine shrimp nauplii (Artemia sp.)
- Feeding frequency: Once or twice a day at a rate sufficient to promote active reproduction and maintain body condition

F0 GENERATION
- Type of food: Commercial flake food (Sera Vipan) and brine shrimp nauplii (Artemia sp.)
- Feeding frequency: Twice each day at a rate sufficient to promote active reproduction and maintain body condition.

F1 GENERATION
- The first feeding began when greater than 90% of the negative control individuals hatched in the second generation
- Type of food for newly hatched larvae: Brine shrimp nauplii (Artemia sp.)
- Feeding frequency of newly hatched larvae: Three times per day during the first seven days post-hatch
- Type of food for F1 generation fish: Brine shrimp nauplii (Artemia sp.) and commercial flake food (from day 2 post hatch)
- Feeding frequency of F1 generation fish: three times daily on weekdays and at least twice daily on weekends and holidays

F2 GENERATION
- No feeding

OTHER FEEDING INFORMATION
- The fish were not fed for at least 12 hours prior to test termination to allow for clearance of the digestive tracts before terminal weight measurements were made.
- Feeding rates in each replicate were adjusted weekly, if necessary, to account for mortality.
- Uneaten food and faecal material were removed from the test vessels daily by siphoning.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

256 d

Remarks on exposure duration:

F0 generation: 21 days exposure; F1 generation: 235 days exposure (196 days prior to formation of reproductive groups, 39 days reproductive phase)

Post exposure observation period:

F0 generation: 14 day Recovery (negative control and high treatment group only); F1 generation: 39 day Recovery; F2 generation: 5 days

Hardness:

- Negative control: 139 ± 6 mg/L (measured as CaCO3)
- Highest concentration (558 g/L): 140 ± 5 mg/L (measured as CaCO3)

Test temperature:

23.8 - 28.0°C

pH:

7.7 - 8.4

Dissolved oxygen:

5.0 - 8.2 mg/L

Conductivity:

- Negative control: 362 ± 27 µS/cm
- Highest concentration (558 g/L): 362 ± 27 µS/cm

Nominal and measured concentrations:

- Nominal concentrations: Negative control, 9, 26, 78, 233 and 700 µg/L
- Mean measured concentrations: < LOQ, 7.9, 21, 63, 188 and 558 µg/L, respectively.

Details on test conditions:

TEST VESSELS
- During all generations the delivery system and test vessels were placed in a temperature controlled environmental chamber to maintain the target water temperature throughout the test period. During the F0 generation and F1 reproductive phase (exposure and recovery), three spawning substrates were maintained in each test vessel during the exposure period. A spawning substrate consisted of an inverted semi-circular section of PVC pipe (suitably leached in dilution water), approximately 10 cm in length placed on a stainless steel tray with a stainless steel mesh false bottom. The test vessels were randomly positioned by treatment group in the environmental chamber and were labelled with the project number, test concentration and replicate designation.
- The aquearia information, water volumes, water depth, flow rate, stock concentration and number of turnovers in the test vessels in different generations during the study can be found in Table 1 in the "Any other information on materials and methods incl. tables"

LOADING
- In order to estimate the maximum loading rates during the test, the loading rate was based on the F1 adult fish weights at thinning on Day 196 post hatch. Each F1 generation test vessel held a maximum of 40 fish prior to thinning on Day 196. Although the ratio of males to females in each replicate varied, the assumption of a 50:50 ratio of males to females among the 40 fish in each replicate was used for the calculation. Biomass loading then was calculated based on the mean wet weight of males and females in the negative control group. The biomass loading was calculated to be 0.36 g per liter of test solution that passed through the test vessel during a 24-hour period. Instantaneous loading (the total wet weight of fish per liter of water in the tank) was 2.2 g of fish per liter of test solution. Loading rates were below the maximum allowable biomass loading rate of 0.5 g/L and the instantaneous loading rate of 5.0 g/L.

LIGHTING:
- Type: Fluorescent light bulbs that emit wavelengths similar to natural sunlight were used to illuminate the test systems.
- Duration: 16 hours/8 hours (light/dark, with a 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting)
- Average light intensity: 759 ± 226 lux (measured weekly during the exposure period in five indiscriminately selected locations within the environmental chamber)

F0 EXPOSURE AND RECOVERY
- An 8-day pre-exposure period immediately preceded the 21-day F0 generation exposure period to identify groups of actively spawning fish.
- No. of organisms per vessel: 6 ( two males and four females)
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- On day 21 of the F0 generation the negative control group and any exposure treatment groups that exhibited a statistically significant effect on egg production, fertility and/or spawning frequency in comparison to the negative control group were transferred to dilution water only for a 14 day recovery assessment period. The remaining treatment levels of the F0 generation that were not moved into recovery were maintained under exposure conditions until it was determined that the initiation of the F1 generation was successful.
- Effect parameters mesured: Survival, fecundity, fertility and general observations of behaviour and health were noted daily during both the exposure and recovery phases. After termination of each phase, lengths and weights of surviving fish were measured and blood samples were collected and stored.

F1 EXPOSURE
- The F1 generation was exposed as embryos and was initiated using eggs from the F0 generation exposure phase. Starting on day 18 of the F0 generation, spawns with >50 eggs were collected and pooled within each treatment for incubation, and when possible, groups of offspring (F1 generation) were reared to adulthood. On day 214 of the study (day 196 post hatch), F1 generation fish were thinned to form reproductive groups.
- No. of organisms per vessel: 6 ( two males and four females )
- No. of vessels per concentration: 2 (one vessel was maintained under exposure conditions and the other vessel was used for recovery)
- No. of vessels per control: 2 (one vessel was maintained under exposure conditions and the other vessel was used for recovery)
- Effect parameters mesured: Hatching success, survival, and general observations of behaviour and health were noted daily during the F1 generation. Growth (photographic length) of F1 generation fish was evaluated periodically throughout the test period. Sex ratio was also evaluated in the F1 generation by histological gonadal sex determination. Egg production and spawning frequency were monitored throughout the reproductive period. Fertility was also assessed on a subset of spawns during the F1 generation. After termination of the reproductive groups, lengths and weights of surviving fish were measured and secondary sex characteristics (including tubercle scores and fatpad appearance) were evaluated. Fish were then processed for gonadal and liver histopathology and plasma vitellogenin (VTG) analyses.

F1 RECOVERY
- On day 215 of the study (day 197 post hatch), one of the F1 exposure vessels formed from the control and each treatment group was transferred to dilution water only and maintained as the recovery phase.
- Effect parameters mesured: Egg production and spawning frequency were monitored throughout the reproductive period. Fertility was also assessed on a subset of spawns during the F1 generation. After termination of the reproductive groups, lengths and weights of surviving fish were measured and secondary sex characteristics (including tubercle scores and fatpad appearance) were evaluated. Fish were then processed for gonadal and liver histopathology and plasma vitellogenin (VTG) analyses. Processed samples (e.g. liver histopathology slides) were stored if not used for immediate analysis.

F2 EXPOSURE AND RECOVERY
- The F2 generation was initiated using eggs from the F1 generation exposure and recovery phases. Starting on Day 218 of the study (day 200 post hatch of the F1 generation), spawns with >50 eggs were collected and pooled within each treatment for incubation.
- No. of organisms per vessel: 2
- No. of vessels per concentration: 2
- No. of vessels per control: 2
- Effect parameters mesured: Each spawn was reared until hatch and then terminated.

Reference substance (positive control):

no

Key result

Duration:

256 d

Dose descriptor:

NOEC

Effect conc.:

188 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

- An overview of the results is provided in the tables in ‘Any other information on results incl. tables’.
- Details of F2 generation results see "Any other information on results inclu. tables".

F0 GENERATION
Survival and General Observations
- There were no significant, treatment-related effects on survival of fathead minnows during the F0 generation. The mean percent survival on Day 21 in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 100, 100, 95.8, 100, 95.8 and 100%, respectively. There were no statistically significant decreases in survival in any treatment group in comparison to the negative control during the F0 generation exposure according to both Jonckheere-Terpstra trend test and Dunnett’s test (p > 0.05).
- During the exposure phase, all surviving fathead minnows in the negative control and all treatment groups appeared normal throughout the test. One male in the 21 µg/L treatment group was observed to have an abrasion along the length of the abdomen on Days 13 to 14 of exposure, and bruising along the side of the abdomen on Day 15. From Day 16 no further observations were noted. All fish appeared normal and healthy in all other respects.
- During the recovery phases, all surviving fathead minnows in the negative control and all treatment groups appeared normal throughout the test.

Reproduction in the 21 Day F0 Generation Exposure
- There were no statistically significant effects on reproduction during the exposure phase with the exception of the high (558 µg/L) treatment group.
- Cumulative eggs produced - There were no effects on the cumulative number of eggs produced with the exception of the high (558 µg/L) treatment group. The mean cumulative number of eggs produced over the 21-day F0 generation exposure in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 3705, 3423, 3666, 3577, 2896 and 1525 eggs, respectively. The decrease in reproduction in the 558 µg/L treatment group was statistically significant in comparison to the negative control according to both Jonckheere-Terpstra trend test and Dunnett’s test (p ≤ 0.05)
- Eggs per Female Reproductive Day - There were no effects on the number of eggs produced per female per day with the exception of the high (558 µg/L) treatment group. The mean number of eggs per female reproductive day in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 44.1, 40.8, 43.6, 42.6, 34.5 and 19.1 eggs per day, respectively. The decrease in reproduction in the 558 µg/L treatment group was statistically significant in comparison to the negative control according to both Jonckheere-Terpstra trend test and Dunnett’s test (p ≤ 0.05).
- Fertility - There were no effects on fertility in this study. The mean percent fertility in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 98.9, 97.7, 98.3, 98.3, 98.1 and 96.6% respectively. There was a significant trend in the 558 µg/L treatment group according to the Jonckheere-Terpstra trend test (p ≤ 0.05); however, there were no statistically significant differences in percent fertility in any treatment groups in comparison to negative control according to Dunnett’s test (p > 0.05).
- Spawning Frequency - There were no effects on spawning frequency with the exception of the high (558 µg/L) treatment group. The mean percent spawning frequency in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 60.7, 53.6, 58.3, 67.9, 53.6 and 27.4%, respectively. The decrease in spawning frequency in the 558 µg/L treatment group was statistically significant in comparison to the negative control according to both Jonckheere-Terpstra trend test and Dunnett’s test (p ≤ 0.05).

Reproduction in the 14 Day F0 Generation Recovery
- When exposure was discontinued, fish previously exposed to 558 µg/L demonstrated recovery from the reproductive effect apparent during exposure. Within one to two days of the start of the recovery phase, egg production in the 558 µg/L treatment recovery group exceeded that of the negative control and continued to exceed the control group production for the remainder of the recovery phase.
- Cumulative Eggs Produced - The mean cumulative number of eggs produced over the 14-day F0 generation recovery phase in the negative control and 558 µg/L treatment recovery group was 2787 and 4059 eggs, respectively. There were no statistically significant decreases in the mean cumulative number of eggs produced in comparison to the negative control (p > 0.05) according to Dunnett’s test.
- Eggs per Female Reproductive Day - The mean number of eggs per female reproductive day in the negative control and 558 µg/L treatment recovery group was 49.8 and 72.5 eggs per day, respectively. There were no statistically significant decreases in the mean cumulative number of eggs per female reproductive day in comparison to the negative control (p > 0.05) according to Dunnett’s test.
- Fertility - The mean percent fertility in the negative control and 558 µg/L treatment recovery group was 98.4 and 98.6% respectively. There were no statistically significant differences in the mean percent fertility in comparison to the negative control (p > 0.05) according to Dunnett’s test.
- Spawning Frequency - The mean percent spawning frequency in the negative control and 558 µg/L treatment recovery group was 64.3 and 71.4%, respectively. There were no statistically significant differences in the mean percent spawning frequency in comparison to the negative control (p > 0.05) according to Dunnett’s test.

F1 GENERATION
Hatch, Survival and General Observations
- There were no effects on hatching success observed at any of the concentrations tested over the five-day hatching period. All embryos hatched on Days 4 and 5 of the F1 generation. Hatching success in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 100, 100, 99.5, 99.5, 100 and 99.5%, respectively (See Table 5 at "Any other information on results incl. tables"). A Fisher’s Exact test indicated that there were no statistically significant decreases in hatching success
between the negative control and any of the treatment groups (p > 0.05).
- Day 23 (Day 0 post-hatch) - There were no treatment-related effects on survival of larval fish that were released on Day 23 of the study from incubation cups into the test vessels. Survival percentages at Day 23 in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 99.5, 99.5, 98.0, 100, 99.5 and 100%, respectively.
- Day 54 and 81 (Day 28 and 56 post-hatch) - Survival also was assessed on Day 54 of the study (approximately Day 28 post-hatch) and Day 81 of the study (approximately Day 56 post-hatch) since fish were photographed for length measurements on those days. There were no effects on survival from hatching until Day 54 of the study or Day 81 of the study. The percentages of survival in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 98.1, 99.4, 100, 98.1, 100 and 99.4% for Day 54 and 98.1, 99.4, 100, 98.1, 99.4 and 99.4% for Day 81, respectively. A Fisher’s Exact test indicated that there were no statistically significant decreases in survival between the negative control and any of the treatment groups (p > 0.05).
- Day 214 (Thinning at Day 192 post-hatch) - There were no treatment-related effects on survival of F1 generation juvenile fish on Day 214 of the test (Day 192 post-hatch) when fish were thinned for the formation of reproductive groups. The percentages of survival in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 97.5, 98.1, 95.6, 96.9, 99.4 and 98.1%, respectively. A Fisher’s Exact test indicated that there were no statistically significant decreases in survival between the negative control and any of the treatment groups (p > 0.05). The majority of fish in the control and all treatment groups of the F1 generation appeared normal throughout the test. Occasional observations of weak or small fish, or fish with morphological deformities such as crooked tails or an injury, were observed in the treatment groups. However, these observations were relatively few in number, were comparable to observations in the control groups, and were not considered to be treatment-related.
- Day 235 (Termination of Exposure Phase Reproductive Groups) - There were no effects on survival at termination of the reproductive groups on Day 253 (Day 235 post-hatch). The percentages of survival in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 100, 100, 95.8, 100, 100 and 100%, respectively. A Fisher’s Exact test indicated that there were no statistically significant decreases in survival between the negative control and any of the treatment groups (p > 0.05).
- Day 234 (Termination of Recovery Phase Reproductive Groups) - There were no effects on survival at termination on Day 252 (Day 234 post-hatch). The percentages of survival in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 100, 95.8, 100, 95.8, 95.8 and 100%, respectively. A Fisher’s Exact test indicated that there were no statistically significant decreases in survival between the negative control and any of the treatment groups (p > 0.05). The majority of fish in the control groups and in all treatment groups of both the exposure and recovery phase reproductive groups appeared normal throughout the test. Occasional observations of enlarged abdomens, egg-bound females or injury, were observed in the treatment groups. However, these observations were relatively few in number, were comparable to observations in the control groups, and were not considered to be treatment related.

Length and Weight
- Day 54 and 81 (Day 28 and 56 post-hatch) - Photographic length measurements were assessed on Days 50 to 54 (Day 28 post-hatch) and Days 78 to 82 (Day 56 post-hatch) of the study to evaluate growth of larval fish.
- The mean lengths in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 20.9, 22.4, 22.2, 22.4, 23.2 and 22.0 mm for Day 54 of the study. The increases in length in all treatment groups from hatching until Day 54 of the study statistically significant according to Dunnett’s Test (p ≤ 0.05).
- The mean lengths in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 34.7, 37.1, 35.8, 36.6, 36.6 and 35.0 mm for Day 82. The Jonkheere-Terpstra trend test demonstrated that there were no significant trends in growth measurements in any of the treatment groups (p > 0.05) on Day 81. The increases in length in the 7.9, 21, 63, and 188 µg/L treatment groups was statistically significant according to Dunnett’s Test (p < 0.05). Although the increases in length were statistically significant on both days 54 and 81 of the study, it was not dose responsive, the increase was slight ranging from 1 to 7% and there was no comparable increase in weight. In addition this variation in length is considered, within this testing laboratory, to be within the range of normal biological variability. Furthermore, at this phase of the study the sex ratio of the fish was not known resulting in the possible biasing of length data at these two time points with sexes essentially being pooled. Therefore, the increases in length on Days 54 and 81 were not considered to be treatment related or of biological significance.
- Day 214 (Thinning at Day 192 post-hatch) - Female Growth - The mean lengths of females in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 48.5, 49.2, 46.3, 47.8, 51.1 and 51.2 mm, respectively. Jonkheere-Terpstra trend test (p ≤ 0.05) detected a significant trend at 558 μg/L. The increase in length in the 188 and 558 µg/L treatment groups was statistically significant according to Dunnett’s test (p ≤ 0.05). However, the increase was slight (~6%) and considered to be within the range of normal biological variability. There was also no dose response between 188 and 558 µg/L and no significant effect or dose response was noted in the corresponding weight data. Therefore it was not considered a treatment-related effect. The mean weights of females in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 1.31, 1.29, 1.11, 1.31, 1.55 and 1.46 g, respectively. There were no statistically significant effects on female weight in any of the treatment groups according to the Jonkheere-Terpstra trend test (p > 0.05) or Dunnett’s test (p > 0.05).
- Day 214 (Thinning at Day 192 post-hatch) - Male Growth - The mean lengths of males in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 54.5, 58.0, 55.2, 55.5, 58.3 and 54.9 mm, respectively. The increase in length in the 7.9 and 188 µg/L treatment groups was statistically significant in comparison to the control according to Dunnett’s test (p ≤ 0.05). However, the increase was slight (~6 to 7%) and considered to be within the range of normal biological variability. Additionally, a Jonkheere-Terpstra trend test demonstrated that there were no significant trends in growth measurements in any of the treatment groups (p > 0.05) for male length and there was no significant trend or difference in the corresponding weight data. Therefore it was not considered a treatment-related effect. The mean weights of males in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 1.94, 2.14, 2.07, 2.03, 2.14 and 1.84 g, respectively. There were no effects on male weight in any of the treatment groups according to the Jonkheere-Terpstra trend test (p > 0.05) or Dunnett’s test (p > 0.05).
- Day 214 (Thinning at Day 192 post-hatch) - Growth of Fish of Undetermined Sex - There was one fish in the 558 µg/L treatment group that was determined to be hermaphroditic when histological sex was evaluated. This fish had a length of 52 mm and a weight of 1.47 g.
- Day 253 (Termination of Exposure Phase Reproductive Groups on Day 235 post-hatch) - Female Growth - The mean lengths of females in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 53.6, 52.2, 49.2, 49.4, 51.7 and 53.6 mm, respectively. The mean weights of females in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 1.70, 1.70, 1.42, 1.60, 1.75 and 2.07 g, respectively. Statistical analyses were not performed due to the limited sample size, four females per replicate, of reproductive groups.
- Day 253 (Termination of Exposure Phase Reproductive Groups on Day 235 post-hatch) - Male Growth - The mean lengths of males in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 62.6, 62.1, 57.5, 61.8, 60.9 and 60.0 mm, respectively. The mean weights of males in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 3.26, 3.50, 3.20, 3.34, 3.38 and 3.53 g, respectively. Statistical analyses were not performed due to the limited sample size, two males per replicate, of reproductive groups.
- Day 253 (Termination of Exposure Phase Reproductive Groups on Day 235 post-hatch) - Growth of Fish of Undetermined Sex (F1 Day 253): There was one fish in the 63 µg/L treatment group that was phenotypically male, however during histological evaluation was determined to be female. This fish had a length of 57 mm and a weight of 3.30 g. This fish was not included in statistical analysis.
- Day 252 (Termination of Recovery Phase Reproductive Groups on Day 234 post-hatch) - Female Growth - The mean lengths of females in the negative control, 7.9, 21, 63, 188 and 558 µg/L recovery groups were 52.9, 51.0, 51.1, 51.8, 52.7 and 54.5 mm, respectively. The mean weights of females in the negative control, 7.9, 21, 63, 188 and 558 µg/L recovery groups were 1.56, 1.51, 1.66, 1.57, 1.67 and 1.98 g, respectively. Statistical analyses were not performed due to the limited sample size of reproductive groups.
- Day 252 (Termination of Recovery Phase Reproductive Groups on Day 234 post-hatch) - Male Growth - The mean lengths of males in the negative control, 7.9, 21, 63, 188 and 558 µg/L recovery groups were 62.3, 63.4, 59.8, 60.5, 63.1 and 61.3 mm, respectively. The mean weights of males in the negative control, 7.9, 21, 63, 188 and 558 µg/L recovery groups were 3.27, 3.71, 3.20, 3.30, 3.63 and 3.29 g, respectively. Statistical analyses were not performed due to the limited sample size of reproductive groups.

Sex Ratio
- The sex of each fish sampled at Day 214, Day 252 and Day 253 was determined by histological examination of the gonads and was combined to evaluate sex ratio for all fish in the F1 generation. There was one fish in the 558 µg/L treatment group on day 214 that was confirmed to be hermaphroditic during histological assessment. At termination of the exposure phase reproductive group, there was one male fish in the 63 µg/L treatment group that was determined to be female during histological assessment. These observations were not considered to be treatment related as it was an isolated to two fish. Fish of undetermined sex were included in the total number of fish when calculating the proportion of fish that were males. There were no treatment-related effects on the sex ratio for the F1 generation of the test. The percentages of male fish from the F1 generation in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 35.4, 61.0, 53.8, 61.8, 51.3 and 52.1%%, respectively. There were no statistically significant differences between the negative control and treatment groups according to both the Jonkheere-Terpstra trend test (p > 0.05) and Dunnett’s test (p > 0.05).

Reproduction in the F1 Generation Exposure Phase
- Cumulative Eggs Produced - The mean cumulative number of eggs produced over the 39-day F1 generation reproductive group exposure in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 5551, 9863, 7425, 9046, 8400 and 1256 eggs, respectively. There was statistically significant (p ≤ 0.05) decrease in cumulative number of eggs produced in the 558 µg/L treatment group in comparison to the negative control according to Dunnett’s test (p ≤ 0.05).
- Eggs per Female Reproductive Day - The mean number of eggs per female reproductive day in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 35.3, 63.2, 48.8, 58.0, 53.8 and 8.8 eggs per day, respectively. There was statistically significant (p ≤ 0.05) decrease in mean number of eggs per female reproductive day in the 558 µg/L treatment group in comparison to the negative control according to Dunnett’s test (p ≤ 0.05). An additional statistical analysis of eggs per female reproductive day in the 558 µg/L
treatment group in comparison to the negative control was performed from Days 225 to 253 of the study. During this period, eggs per female per reproductive day from the control and the former 558 µg/L test substance exposure group were significantly different (p ≤ 0.05).
- Fertility - There were no effects on fertility in this study. The mean percent fertility in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 99.7, 98.4, 95.9, 99.3, 98.0 and 100% respectively. There were no statistically significant differences in percent fertility in any treatment groups in comparison to the negative control (p > 0.05) according to Dunnett’s test.
- Spawning Frequency - There were no effects on spawning frequency with the exception of the high (558 µg/L) treatment group. The mean percent spawning frequency in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 73.7, 81.4, 66.0, 74.4, 75.0 and 20.9%, respectively. The decrease in spawning frequency (71%) in the 558 µg/L treatment group was statistically significant (p ≤ 0.05) in comparison to the negative control according to both the Jonckheere-Terpstra trend test and Dunnett’s test. The decrease in spawning frequency observed at the 558 µg/L treatment group is possibly biased by the delay in the first spawning in this group.

Reproduction in the F1 Generation Recovery Phase
- There were no statistically significant effects on reproduction during the recovery phase with the exception spawning frequency in the 558 µg/L recovery group.
- Cumulative Eggs Produced - There were no effects on the cumulative number of eggs produced. The mean cumulative number of eggs produced over the 39-day F1 generation reproductive group recovery phase in the negative control, 7.9, 21, 63, 188 and 558 µg/L recovery groups was 6051, 9427, 5922, 8278, 7792 and 5755 eggs, respectively. Since additional females (8 in total) were added to the 558 µg/L recovery group at the time of reproductive group formation, cumulative eggs produced were not statistically analysed for the this recovery group. There were no statistically significant differences on the cumulative number of eggs produced in 7.9, 21, 63, and 188 µg/L recovery groups in comparison to the negative control (p > 0.05) according to Dunnett’s test.
- Eggs per Female Reproductive Day - There were no effects on the number of eggs produced per female per day. The mean number of eggs per female reproductive day in the negative control, 7.9, 21, 63, 188 and 558 µg/L recovery groups was 39.8, 63.2, 39.0, 55.5, 51.4, and 25.2 eggs per day, respectively. There were no statistically significant differences on the number of eggs produced per female per day in any recovery groups in comparison to the negative control (p > 0.05) according to Dunnett’s test. An additional statistical analysis of eggs per female reproductive day in the 558 µg/L treatment group in comparison to the negative control from Day 225 to 253 of the study was performed. During this period, Dunnett’s test found that the differences in eggs per reproductive day in the two groups for days 225 to 252 in the recovery groups were not significantly different (p > 0.05). Thus the statistically significant reduction in F1 egg production observed in the exposure reproductive groups was not apparent during the recovery phase.
- Fertility - There were no effects on fertility in this study. The mean percent fertility in the negative control, 7.9, 21, 63, 188 and 558 µg/L recovery groups was 97.7, 98.2, 98.6, 98.8, 98.1 and 97.9% respectively. There were no statistically significant differences in percent fertility in any recovery groups in comparison to the negative control (p > 0.05) according to Dunnett’s test.
- Spawning Frequency - The mean percent spawning frequency in the negative control, 7.9, 21, 63, 188 and 558 µg/L recovery groups was 80.9, 82.9, 59.9, 77.9, 74.3 and 52.8%, respectively. There was a statistically significant trend in the 558 µg/L recovery group according to the Jonckheere-Terpstra trend test (p < 0.05). The decrease in spawning frequency in the 21 and 558 ug/L recovery groups were statistically significant (p ≤ 0.05) in comparison to the negative control according to Dunnett’s test. While there was a significant
decrease in spawning frequency in the 21µg/L recovery group, it was not dose responsive and not considered to be treatment related. In addition the decrease in spawning frequency observed in the 558 µg/L treatment group is possibly biased by the delay in the first spawning in this group.

Secondary Sex Characteristics
- Prominent secondary sex characteristics in male fathead minnows are breeding tubercles on the head and fatpads on the dorsal nape. Tubercle scores are a quantitative count and tend to be a more reliable indicator of effects on secondary sex characteristics. The presence of dorsal fatpads is a subjective observation taken at test termination to help qualitatively evaluate secondary sex characteristics.
- Exposure Phase - There were treatment related effects on the mean tubercle scores among males in the 21, 63, 188 and 558 µg/L treatment groups in comparison to the negative control. The mean tubercle score for males in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 11.0, 10.9, 22.1, 28.1, 34.0, and 36.3, respectively. There was a statistically significant trend in the 21, 63, 188 and 558 µg/L treatment groups according to the Jonckheere-Terpstra trend test (p < 0.05). The increases in tubercle scores in the 21, 63, 188 and 558 µg/L treatment groups were dose responsive, statistically different from the negative control according to Dunnett’s test (p < 0.05) and considered to be treatment related. All males in the control and treatment groups had visible fatpads. There was no apparent difference in size or shape in dorsal fatpads between the treatment groups and the negative control group. No female fish in the test had tubercles or was noted to have a fatpad at test termination. All females in the negative control and treatment groups were noted with visible ovipositors. One fish in the 63 µg/L treatment group was appeared to be phenotypically male, however during histological evaluation it was determined to be histopathologically female. This was not considered to be treatment related as it was isolated to a single fish.
- Recovery Phase - There were treatment related effects on the mean tubercle scores among males in the 21, 63, 188 and 558 µg/L recovery groups in comparison to the negative control. The mean tubercle score for males in the negative control, 7.9, 21, 63, 188 and 558 µg/L recovery groups was 9.5, 13.3, 15.1, 26.8, 32.8 and 33.5, respectively. There was a statistically significant trend in the 63, 188 and 558 µg/L recovery groups according to the Jonckheere-Terpstra trend test (p < 0.05). The increases in tubercle score in the 63, 188 and 558 µg/L recovery groups were dose responsive, statistically different from the negative control according to Dunnett’s test (p < 0.05) and considered to be treatment related. All males in the control and treatment groups had visible fatpads. There was no apparent difference in size or shape in dorsal fatpads between the treatment groups and the negative control group. No female fish in the test had tubercles or was noted to have a fatpad at test termination All females in the negative control and treatment group were noted with visible ovipositors.

Vitellogenin (VTG) in the F1 Generation Exposure Phase
- Males –The mean VTG concentration in males in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 4.93 x 102, 2.10 x 103, 8.41 x 102, 5.15 x 102, 5.00 x 103 and 4.47 x 102 ng/mL, respectively. There was a statistically significant difference in the 7.9 and 188 µg/L treatment groups according to Dunnett’s test (p < 0.05), however the JonckheereTerpstra trend test showed no significant trend (p > 0.05). Any differences in VTG were not dose responsive and therefore not considered to be treatment related.
- Females –The mean VTG concentration in females in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 1.03 x 107, 9.47 x 106, 5.76 x 106, 6.19 x 106, 4.77 x 106 and 4.39 x 105 ng/mL, respectively. The decrease in VTG concentrations in the 188 and 558 µg/L treatment groups were statistically significant according to Dunnett’s test and the Jonckheere-Terpstra trend test (p < 0.05). However VTG is highly variable, and although the slight decrease in VTG in the 188 µg/L treatment group is statistically significant it could be considered to be within the range of biological variability. The decrease in VTG at the 558 µg/L test concentration however was large, outside normal variability and was therefore considered to be treatment related.


Vitellogenin (VTG)in the F1 Generation Recovery Phase
- Males –The mean VTG concentration in males in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 7.09 x 102, 1.63 x 104, 1.76 x 103, 1.41 x 103, 4.15 x 103 and 5.50 x 103 ng/mL, respectively. The decrease in VTG concentration in the 7.9 µg/L treatment group was statistically significant according to Dunnett’s test (p < 0.05), however the Jonckheere-Terpstra trend test showed no significant trend (p > 0.05). Any differences in VTG were not dose responsive and therefore not considered to be treatment related.
- Females – There were no statistically significant differences in female VTG concentrations in any treatment group in comparison to the negative control. The mean VTG concentration in females in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 1.13 x 107, 7.21 x 106, 6.65 x 106, 8.15 x 106, 6.06 x 106 and 6.19 x 106 ng/mL, respectively. There were no statistically significant differences according to both the Jonckheere-Terpstra trend test and Dunnett’s test (p > 0.05). Any differences in VTG were not dose responsive and therefore not considered to be treatment related.

Gonadal Histopathology
- The gonads from a total of 143 fish (47 males and 96 females) from the F1 generation adult reproductive groups exposed to the test substance were studied. Treatment-related findings in the gonads included increased interstitial cells and increased spermatozoa in males, and decreased yolk formation, decreased post-ovulatory follicles, and a decreased mean ovarian stage score in females. Except for increased interstitial cells, treatment-related findings in the gonads were confined to fish of the 558 µg/L dose group. Findings of increased interstitial cells and especially increased spermatozoa in the testes of 558 µg/L treatment group males were consistent with an increase in the mean gonadosomatic index (GSI) value for that group relative to controls. The prevalence of oocyte atresia was comparable between control females and those of the 558 µg/L treatment group, although atresia occurred at a relatively lesser frequency in females of the 7.9, 21, 63, and 188 µg/L treatment groups. The decrease in post-ovulatory follicles in the 188 and 558 µg/L treatment groups relative to controls is consistent with reduced spawning activity, and oocyte retention may account for the increased GSI in females of those groups compared to controls. Mixed sex is an infrequently reported finding for fathead minnows, although ovarian spermatogenesis has been observed in at least two large studies that were conducted using adults of this species (personal experience of the pathologist). In both of those studies, the prevalence of ovarian spermatogenesis was comparable between treated and control fish, and it was therefore considered to be an incidental to treatment. Consequently, the toxicological relevance of mixed sex in the current study is uncertain because this finding occurred in the gonads of only a single individual, albeit a 558 µg/L treatment group fish. Furthermore there was an occurrence of a phenotypically male fish in the 63 µg/L treatment group with histopahtologically female gonadal tissue.

Liver Histopathology
- The livers from a total of 72 fish (24 males and 48 females) from the adult reproductive groups exposed to the test substance were studied. Treatment-related effects in the livers of test substance-exposed fish included a variety of changes that are consistent with liver cell loss or damage (hepatocellular necrosis, cystic degeneration, and the presence of pigmented macrophage aggregates) and liver regeneration (hepatocyte nuclear pleomorphism, hepatocyte anisocytosis, and increased numbers of multinucleated hepatocytes). Although cystic degeneration and pigmented macrophage aggregates may also occur spontaneously or constitutively in the livers of fish (Wolf and Wolfe, 2005), neither of these are typically observed as background findings in P. promelas, and they were not present in
controls. The relative decrease in hepatic basophilia in females of the 558 µg/L treatment group is indicative of decreased vitellogenin production, as basophilia in the livers of reproductively active female fish is considered to be a morphologic manifestation of unregulated vitellogenin mRNA expression. Based on the degree of damage and repair evident in the livers of some 558 µg/L treatment group females, it is plausible that decreased vitellogenin production may have been the result of functional liver impairment caused by hepatotoxicity. However, because hepatic generation of vitellogenin is a response dependant on numerous other factors, it is not possible to confirm the cause of the VTG reduction.

Reported statistics and error estimates:

See the statistical tests in "Any other information on materials and methods incl. tables"

F2 GENERATION

Hatch, Survival and General Observations in the Exposure Phase

- There were no effects on hatching success observed at any of the concentrations tested over the five-day hatching period in either of the two F2 generation spawns that were initiated in the exposure phase. All embryos hatched on Days 4 to 6 of the test. Hatching success of the first spawn of the exposure phase in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 99.0, 99.5, 100, 100, 99.0 and 98.0%, respectively. Hatching success of the second spawn of the exposure phase in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 100, 98.5, 99.0, 99.5, 99.5 and 95.5%, respectively. A Fisher’s Exact test indicated that there were no statistically significant decreases in hatching success between the negative control and any of the treatment groups (p > 0.05).

- Day 5 (Day 0 post-hatch) - There were no treatment-related effects on survival of larval fish that were released on Day 5 of the test from incubation cups into the test vessels in either of the two F2 generation spawns that were initiated in the exposure groups. Survival percentages at release of the first spawn from the incubation cups in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 100, 99.5, 100, 100, 100 and 100%, respectively. Survival percentages at release of the second spawn from the incubation cups in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 97.5, 100, 94.6, 97.5, 100 and 97.4%, respectively. A Fisher’s Exact test indicated that there were no statistically significant decreases in survival between the negative control and any of the treatment groups (p > 0.05). The majority of fish in the control and 7.9, 21, 63, and 188 µg/L treatment groups of the F2 generations in the exposure phase appeared normal throughout the F2 generation exposure period. Observations of weak fish, fish lying on the bottom, fish with morphological deformities such as crooked tails or fish with an injury were observed in the treatment groups, but these observations were comparable to the control groups, and were not considered to be treatment-related. Observations of fish lying on the bottom, and fish with morphological deformities such as crooked or curled tails or an injury appeared to increase in the 558 µg/L treatment group in comparison to the control.

 

Hatch, Survival and General Observations in the Recovery Phase

- There were no effects on hatching success observed at any of the concentrations tested over the five-day hatching period in either of the two F2 generation spawns that were initiated in the recovery phase. All embryos hatched on Days 4 and 5 of the test. Hatching success of the first spawn of the recovery phase in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 99.5, 99.5, 98.0, 99.5, 99.0 and 99.5%, respectively. Hatching success of the second spawn of the recovery phase in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups was 99.5, 100, 100, 99.5, 100 and 99.5%, respectively. A Fisher’s Exact test indicated that there were no statistically significant decreases in hatching success between the negative control and any of the treatment groups (p > 0.05).

- Day 5 (Day 0 post-hatch) - There were no treatment-related effects on survival of larval fish that were released on Day 5 of the test from incubation cups into the test vessels in either of the two F2 generation spawns that were initiated in the recovery phase. Survival percentages at release of the first spawn from the incubation cups in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 100, 100, 100, 100, 99.5 and 100%, respectively. Survival percentages at release of the second spawn from the incubation cups in the negative control, 7.9, 21, 63, 188 and 558 µg/L treatment groups were 100, 99.5, 100, 100, 100 and 99.5%, respectively. A Fisher’s Exact test indicated that there were no statistically significant decreases in survival between the negative control and any of the treatment groups (p > 0.05).

- The majority of fish in the control and treatment groups of the F2 generation appeared normal throughout the F2 generation recovery period. Observations of weak fish, fish lying on the bottom, fish with morphological deformities such as crooked tails or fish with an injury were observed in the treatment groups, but these observations were comparable to the control groups, and were not considered to be treatment-related.

Table 2. F0 Generation - Summary of Survival, Fecundity, Fertility and spawning Frequency after 21 Day Exposure to the test substance

Mean Measured Concentration (µg/L)	Percent Survival to Termination (Mean ± Std. Dev.)	Cumulative Number of Eggs Produced (Mean ± Std. Dev.)	Eggs per Female per Reproductive Day (Mean ± Std. Dev.)	Percent Fertility (Mean ± Std. Dev.)	Percent Spawning Frequency (Mean ± Std. Dev.)
Negative Control	100 ± 0.0	3705 ± 323	44.1 ± 3.88	98.9 ± 0.3	60.7 ± 9.8
7.9	100 ± 0.0	3423 ± 1041	40.8 ± 12.42	97.7 ± 1.9	53.6 ± 13.7
21	95.8 ± 8.4	3666 ± 773	43.6 ± 9.21	98.3 ± 0.5	58.3 ± 4.6
63	100 ± 0.0	3577 ± 810	42.6 ± 9.62	98.3 ± 0.7	67.9 ± 7.1
188	95.8 ± 8.4	2896 ± 512	34.5 ± 6.13	98.1 ± 0.8	53.6 ± 7.1
558	100 ± 0.0	1525 ± 1078*^†	19.1 ± 13.49*^†	96.6 ± 2.8*	27.4 ± 18.4*^†

* Statistically significant trend using the Jonckheere-Terpstra trend test (p≤ 0.05).
†Statistically significant difference from the negative control using Dunnett’s test (p≤ 0.05)

Table 3. F0 Generation - Summary of Survival, Fecundity, Fertility and Spawning Frequency after 14 Day Recovery Phase

Mean Measured Concentration (µg/L)	Percent Survival to Termination (Mean ± Std. Dev.)	Cumulative Number of Eggs Produced (Mean ± Std. Dev.)	Eggs per Female per Reproductive Day (Mean ± Std. Dev.)	Percent Fertility (Mean ± Std. Dev.)	Percent spawning Frequency (Mean ± Std. Dev.)
Negative Control	100 ± 0.0	2787 ± 239	49.8 ± 4.23	98.4 ± 0.6	64.3 ± 15.4
588	100 ± 0.0	4059 ± 822	72.5 ± 14.70	98.6 ± 0.8	71.4 ± 17.5

Table 4. F1 Generation - Summary of Hatching Success and Survival During Exposure to the test substance

Mean Measured Concentration (µg/L)	Percent Hatching Success^1 (%)	Percent Larval Survival to Thinning on Day 0 Post-Hatch (%)	Percent Survival to Day 28 Post-Hatch (%)	Percent Survival to Day 56 Post-Hatch (%)	Percent Survival to Thinning on Day 196 Post-Hatch (%)	Percent Survival to Termination on Day 235 Post-Hatch^1 (%)	Percent Survival to Termination on Day 234 Post-Hatch^2 (%)
Negative Control	100	99.5	98.1	98.1	97.5	100	100
7.9	100	99.5	99.4	99.4	98.1	100	95.8
21	99.5	98.0	100	100	95.6	95.8	100
63	99.5	100	98.1	98.1	96.9	100	95.8
188	100	99.5	100	99.4	99.4	100	95.8
558	99.5	100	99.4	99.4	98.1	100	100

¹Terminaton of the adult reprodcution exposure groups.

²Termination of the adult reproduction recovery groups.

Table 5. F1 Generation - Summary of Fecundity, Fertility and Spawning Frequency of the Exposure Phase

Mean Measured Concentration (µg/L)	Cumulative Number of Eggs Produced (Mean ± Std.Dev.)1	Eggs per Female per Percent FertilityPercent Spawning Frequency (Mean ± Std.Dev.)^1	Precent Fertility (Mean ± Std. Dev.)	Precent Spawning Frequency (Mean ± Std. Dev.)
Negative Control	5551 ± 2402	35.3 ± 5.4	99.7 ±0.5	73.7 ± 5.7
7.9	9863 ± 1114	63.2 ± 7.1	98.4 ± 1.3	81.4 ± 8.2
21	7425 ± 264	48.8 ± 4.0	95.9 ± 2.5	66.0 ± 16.7
63	9046 ± 313	58.0 ± 2.0	99.3 ± 1.4	74.4 ± 2.1
188	8400 ± 363	53.8 ± 2.3	98.0 ± 1.4	75.0 ± 5.3
558	1256 ± 1421^†	8.5 ± 9.6^†	100	20.9 ± 13.3*^†

* Statistically significant trend according to the Jonkheere-Terpstra trend test (p< 0.05).
† Statistically significant difference in comparison to the negative control according to Dunnett’s test (p< 0.05).
1The data failed the assumption of homogeneity of variances and normality and were subjected to Box-Cox transformation (with Lambda=1.25) to correct the condition.

Table 6. F1 Generation - Summary of Fecundity, Fertility and Spawning Frequency of the Recovery Phase

Mean Measured Concentration (µg/L)	Cumulative Number of Eggs Produced (Mean ± Std. ev.)^1	Eggs per Female per Reproductive Day (Mean ± Std. Dev.)^1	Precent Fertility (Mean ± Std. Dev.)	Precent Spawning Frequency (Mean ± Std. Dev.)
Negative Control	6051 ± 764	39.8 ± 5.1	97.7 ± 1.9	80.9 ± 5.8
7.9	9427 ± 1589	63.2 ± 8.6	98.2 ± 0.7	82.9 ± 3.4
21	5922 ± 2607	39.0 ± 17.1	98.6 ± 2.3	59.9 ± 18.8†
63	8278 ± 2799	55.5 ± 19.4	98.8 ± 1.1	77.6 ± 5.5
188	7792 ± 698	51.4 ± 4.4	98.1 ± 2.4	74.3 ± 3.9
558	5755 ± 2788	25.2 ± 12.2	97.9 ± 1.5	52.8 ± 8.2*†

* Statistically significant trend according to the Jonkheere-Terpstra trend test (p< 0.05).
† Statistically significant difference in comparison to the negative controls according to Dunnett’s test (p< 0.05).
1The data failed the assumption of homogeneity of variances and normality and were subjected to Box-Cox transformation (with Lambda=1.25) to correct the condition.

Table 7. F2 Generation - Summary of Hatching Success During the Exposure and Recovery Phases

	Exposure Phase				Recovery Phase
	Spawn 1		Spawn 2		Spawn 1		Spawn 2
Mean Measured Concentration (µg/L)	Percent Hatching Success (%)	Percent Larval Survival to Day 0 Post- Hatch (%)	Percent Hatching Success (%)	Percent Larval Survival to Day 0 Post- Hatch (%)	Percent Hatching Success (%)	Percent Larval Survival to Day 0 Post- Hatch (%)	Percent Hatching Success (%)	Percent Larval Survival to Day 0 Post- Hatch (%)
Negative Control	99.0	100	100	97.5	99.5	100	99.5	100
7.9	99.5	99.5	98.5	100	99.5	100	100	99.5
21	100	100	99.0	94.6	98.0	100	100	100
63	100	100	99.5	97.5	99.5	100	99.5	100
188	99.0	100	99.5	100	99.0	99.5	100	100
558	98.0	100	95.5	97.4	99.5	100	99.5	99.5

Validity criteria fulfilled:

yes

Remarks:

See the validity criteria in "Any other information on materials and methods incl. tables"

Conclusions:

Based on mean measured concentrations and effects on reproductive endpoints, the NOAEC for the test substance was 188 µg/L.

Executive summary:

Three generations of fathead minnows (Pimephales promelas) were exposed to a geometric series of five test concentrations of the test substance and a negative control (dilution water) under flow through conditions. The fish were exposed to the test substance at the mean measured concentrations of 7.9, 21, 63, 188 and 588 µg/L (measured by LC/MS/MS; the nominal concentrations were 9.0, 26, 78, 233, 700 µg/L, respectively). Parental generation (F0) fish were proven breeders exposed as adults in reproductive groups for three weeks. Egg production, fertility and spawning frequency were monitored during the parental generation exposure period. Starting after 18 days of exposure of the F0 generation, where produced eggs were collected to initiate the F1 generation. Due to the lack of reproduction in the high treatment group, the F1 generation at this concentration was initiated using eggs from the negative control. At the end of the 21 day F0 generation exposure, the control group and any treatment group that showed a statistical difference from the control were moved to a 14 day recovery phase in untreated dilution water. The F1 generation was raised to adulthood and on Days 214 and 215 of the study (196 and 197 days post-hatch), the F1 generation fish were thinned to form two reproductive groups from each replicate in the control and treatment groups. One reproductive group was exposed to the test substance until the end of the study, while the other was moved into a recovery phase consisting of dilution water only. Eggs produced by the F1 generation in both exposure and recovery phases were collected at suitable time points to initiate the F2 generation and groups of offspring (F2 generation) were reared until day 5 post hatch. The F0 generation was monitored for survival and reproduction. The F1 generation was monitored for hatching success, survival, growth (length and weight), reproduction and sex ratio (by histological confirmation of sex). Reproductive fish from the F1 generation fish were also evaluated for blood plasma vitellogenin (VTG) levels and gonadal and liver histopathology. The F2 generation was monitored for hatching success and survival.

F0 Generation:
There were no effects on survival at any concentrations. Effects on reproductive parameters were restricted to the highest concentration (558 μg/L) where a decrease in cumulative eggs, eggs/female/day and spawning frequency were observed. No effects on fertility were observed in any concentrations. The effects observed in highest concentration returned to levels comparable with the control fish shortly after moving the fish into the recovery phase.

F1 Generation:
Survival and hatch of the F1 generation was not affected at any concentration tested throughout the F1 generation. Statistically significant effects in length were observed in all concentrations at various time points throughout the study. Although the increases in length were statistically significant, it was not dose responsive, the increase was light ranging from 1 to 7% and there was no comparable increase in weight. In addition this variation in length is considered, within this testing laboratory, to be within the range of normal biological variability.No effects were observed on sex ratio at any concentration tested.Statistically significant reproductive effects (cumulative number of eggs,eggs/female/day, and spawning frequency) were observed at the highest tested concentration (558 μg/L).No effects on fertility were observed at any concentration tested.
Additional treatment related effects include increase in male tubercle score at and above concentrations of 21 µg/L and statistically significant reduction of vitellogenin in females at 558 µg/L. Histopathological effects in the gonads and liver of the F1 Generation (Day 235) were:

- Interstitial cell hyperplasia / hypertrophy and increased spermatozoa in the testes of males
- Decreased yolk formation, decreased post-ovulatory follicles, and decreased mean ovarian stage scores in the ovaries of females
- Hepatocyte nuclear pleomorphism, hepatocyte anisocytosis, hepatocellular necrosis, increased numbers of multinucleated hepatocytes, cystic degeneration, and pigmented macrophage aggregates in the livers of both sexes
- Reduced hepatic basophilia in the livers of females only

The lowest concentration at which histological effects were observed (interstitial cell hyperplasia / hypertrophy in males) was 63 µg/L.

F2 Generation:
Hatching and survival of the F2 generation was unaffected at all concentrations tested.

Based on the findings, the NOAEC for the test substance was 188 µg/L.

Description of key information

 All available data was assessed and the studies representing the worst-case effects were included as key or weight-of-evidence studies. Other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA.  

Freshwater; 256-d NOEC = 0.188 mg/L; reproduction; Pimephales promelas; OECD TG 229; Schneider, 2014

Marine water; 100-d NOEC = 0.068 mg/L; reproduction; Cyprinodon variegatus; EPA TG 72 -5; Breteler, 1988

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

0.188 mg/L

Marine water fish

Marine water fish

Dose descriptor:

NOEC

Effect concentration:

0.068 mg/L

Additional information

Three studies on freshwater fish and one study on marine water fish are available for this endpoint. The 256-d study on fathead minnows (Pimephales promelas) (Schneider et al. 2014, OECD TG 229, GLP, Reliability 1) and 100-d study on sheepshead minnow (Cyprinodon variegatus) (Breteler 1988, EPA 72-5, GLP, Reliability 2) were selected as key studies because they represent the worst-case effects (i.e. showed the lowest E(L)C50 value) for freshwater and marine water conditions, respectively. 

In the freshwater key study, fathead minnows (Pimephales promelas) were exposed to the test substance at nominal concentrations of 9.0, 26, 78, 233, 700 µg/L (mean measured concentrations of 7.9, 21, 63, 188 and 588 µg/L, respectively) in a flow-through system. The conditions of the test were as follows: temperature of 24 - 28°C, pH 7.7 – 8.4 and dissolved oxygen 5.0 – 8.2 mg O2/L. The results show that the NOEC for reproduction was 0.188 mg/L based on mean measured concentrations. The two supporting studies on freshwater fish show that NOECs ranging from 0.31 – 0.43 mg/L, based on mean measured concentrations (Rufli 1994 and Surprenant 1987).

In the marine water key study, sheepshead minnow were exposed to the test substance at nominal concentrations of 0.019, 0.038, 0.075, 0.15, 0.30 and 0.60 mg/L (measured concentrations of 0.016, 0.038, 0.068, 0.15, 0.29 and 0.55 mg/L, respectively). The number of animals were as follows: days 0 - 5 F0 200 eggs/dose level; days 5 - 32 F0 100 larvae/dose level; days 32 - 52 F0 50 juvenile/dose level; days 52 - 85 F0 50 adults/dose level; days 55 - 100 F1 100/dose level. The conditions of the test were as follows: salinity 30 – 32‰, temperature of 27 – 31°C, pH 7.9 – 8.2 and dissolved oxygen 64 – 70 % of saturation for 14 days. The results show that the NOEC for reproduction was 0.068 mg/L based on mean measured concentrations.