Chlorothalonil

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:

31 May 1978 to 26 Mar 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted in according to EPA "Recommended bioassay procedures for fathead minnow (Pimephales promelas, Rafinesque) chronic tests" (1971).

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Water was sampled weekly from each duplicate aquarium and analyzed for the test substance concentrations. On the last sampling day of each month, water samples were also analyzed for the metabolite of the test substance. On test days 62, 141 and 167, a water sample was collected from the delivery tube in each aquarium in the A replicate, in addition to the aquarium sample, to determine if influent concentrations of the test substance were comparable to aquaria concentrations. Water samples collected on days 62 and 141 represented one diluter cycle, while the day 167 samples represent three diluter cycles.

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

- Stock solutions were prepared on a weight: volume basis every two weeks by dissolving 0.7208 g of the test substance with analytical grade acetone in a 100 mL volumetric flask. The 100 mL stock solutions were stored in a foil-covered glass bottle at 20°C in a closed cabinet. Samples of the stock solutions were analyzed for the test substance when prepared and two weeks after preparation.

- The solvent control contained 4.5 µL/L acetone which was approximately equal to the amount of acetone present in the highest test concentration (3.6 µL/L). The difference in these solvent concentrations was due to the limitations of the ma diluter injector mechanism.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Age at study initiation: Eggs
- Source: Brood stock from the test facility

FEEDING
- Food type and frequency: Newly-hatched fry were fed live brine shrimp nauplii three times daily for twenty days, after which fish were fed frozen brine shrimp three timesdaily. Live Daphnia magna were substituted for one of the daily feedings of frozen brine shrimp when available.
- Feeding quality: To demonstrate the absence of pesticide and PCB residues, a pesticide scan was performed on a sample of the brine shrimp eggs used during the study. Two samplets of frozen brine shrimp, one from each of the two shipment lots used during the study, were also analyzed. The analytical results revealed these food sources to be free of pesticide or PCB contamination with all results below detectable levels.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

297 d

Hardness:

29 - 31 mg/L as CaCO3

Test temperature:

25 ± 2°C

pH:

6.5 - 7.4

Dissolved oxygen:

- 7.2 - 8.5 mg O2/L
- >60% of saturation

Conductivity:

135 - 146 µmhos/cm

Nominal and measured concentrations:

- Nominal concentration: 0 (negative control), 0 (solvent control), 1.5, 3.1, 6.2, 12 and 25 µg/L
- Measured concentration: < LOQ (negative control), < LOQ (solvent control), 0.6, 1.4, 3.0, 6.5 and 16 µg/L, respectively. See Table 1 in 'Any other information on materials and methods incl tables'

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquarium
- Size of vessel: 90 x 30 x 30 cm
- Subdivision of the vessel: The glass aquarium was subdivided by a stainless steel screen to provide a spawning area (50 x 30 x 30 cm) and an area where egg cups were suspended and two larval growth chambers (30 x 12 x 25 cm) were placed.
- Spawning sites: Five spawning sites were made from 7.5 cm sections (halved) of cementasbestos drain pipe 10 cm in diameter and were placed, concave surface down, in each spawning chamber.
- Egg incubation cup: Egg groups were incubated in cups made from 5 cm diameter glass jars with 40 mesh stainless steel screen bottoms. Egg cups were oscillated a vertical travel distance of 1-1.5 inches in the test water by a rocker arm driven by a 2 rpm motor.
- Filled volume: The water level in each aquarium was maintained at 15 cm by a standpipe.
- Type of flow-through: Proportional diluter
- Dilution factor: 0.50
- Flow rate: 8 times the volume (40 L) per 24 hours.
- No. of vessels per concentration: 2
- No. of vessels per control: 2
- No. of vessels per vehicle control: 2
- Exposure system: A 50 mL gas-tight syringe with a stainless steel needle was activated by a mechanical injector to deliver the toxicant to the glass mixing chamber of the diluter. Polyethylene tubing was attached to the end of the needle and extended below the surface of the water in the mixing chamber where a magnetic stirring bar continuously , mixed the contents. The syringe was refilled with stock solution weekly and covered with black polyethylene to minimize potential light degradation of the test material. A flow-splitting chamber was used to promote further mixing of each dilution of the toxicant prior to one liter of the mixture flowing to duplicate aquaria through separate glass delivery. The flow-splitting chamber over each aquarium directed 700 mL of the test solutions to the spawning area and 150 mL to each of the growth chambers.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The diluent water used in this study was drawn from a 125 meter deep bedrock well into a concrete reservoir where it was aerated and supplemented with well water supplied by the Town of Wareham, Massachusetts. No water was drawn from the thest facility well between test days 269 - 278 during well reclamation operations and 100% of the diluent water was from the town supply. Because of the softness of the water, diluent water was passed through a water hardening cartridge, containing crushed oyster shells, which was attached to the chronic unit. The cartridoe was used from day 267 until the end of the study.
- Dilution water measurment: The diluent water was analyzed at the beginning and near the middle of the study for solids and total dissolved solids.
- Calcium: 4.4 - 9.4 mg/L
- Magnesium: 1.9 - 2.6 mg/L
- Sodium: 13 - 16 mg/L
- Potassium: 1.0 mg/L
- Chloride: 15 - 18 mg/L
- Sulfate: 9.8 ± 0.3 mg/L to 12 ± 2 mg/L
- Total solids: 85 ± 5 mg/L to 111 ± 1 mg/L
- Total dissolved solids: 28 mg/L to 62 ± 4 mg/L
- Aquarium cleaning: Aquaria were syphoned clean of debris twice weekly and brushed when fungus or algae growth began to build up. Diluter cells were brushed weekly and the amount of water being delivered through the system to each aquarium was measured monthly.
- Intervals of water quality measurement: Temperature and dissolved oxygen were measured in each aquarium on a rotating basis, so each aquarium was measured at least once weekly. Total hardness, alkalinity, pH, acidity and specific conductivity were measured weekly in one control aquarium and one treatment level aquarium on a rotating basis

OTHER TEST CONDITIONS
- Photoperiod: The photoperiod followed the EPA recommended schedule using the daylength of Evansville, Indiana on December 1 for the first day of the test and adjusting the daylength on the first and fifteenth day of each month by means of an automatic timer.
- Light intensity and location: Illumination was provided by a combination of Durotest (Optima FS 50) and wide spectrum Grow-Lux fluorescent lights which were centrally located 64 cm above the aquaria water surface. The entire experimental unit was enclosed in black polyethylene curtains to prevent disturbance of the fish and to minimize the interference of laboratory lighting with the intended photoperiod.

EXPOSURE PRCEDURE AND EFFECT PARAMETERS MEASURED:
- The chronic study was initiated by incubating 100 eggs divided between two egg cups in each aquarium. Eggs were randomly assigned (by twos) to each of 28 egg cups until a total of 50 eggs per cup was reached. Each day, until hatching was completed (4 days), the eggs in each cup were counted and dead eggs removed. Percentage hatching success (number of live fry/100 eggs x 100) was calculated for each duplicate aquarium. Twenty newly hatched fry were impartially selected from each egg cup and placed in their respective growth chambers. Each fry group was photographed over a millimeter grid on test days 35 and 64 to determine total lengths and percentage survival. On test day 64, eight fish from one fry chamber and seven fish from the other fry chamber in each duplicate aquarium were impartially selected to continue exposure and were photographed as a group, Extra fish from each duplicate aquarium were photographed as a group, weighed in tared glass vials and frozen for possible residue analysis.

- On test day 155, an attempt was made to determine the sex of each fish in order to establish a sex ratio of three males to six females in each aquarium. After examining the fish in three aquaria, it was decided to postpone the thinning process until sexual characteristics were more developed. On test day 172, the fish were again examined. Three males and six females were retained as spawners in all aquaria except where fewer than six females were present. Terminated fish from each aquarium were pooled and frozen as whole fish for possible residue analysis.

- After day 155, the underside of the spawning tiles were examined daily for eggs. Spawning was very sparse until the sex ratio was adjusted on day 172. Spawns were removed from the tiles by gently rolling the egg mass with the fingertips. The number of eggs in each spawn was recorded and groups of fifty eggs from a maximum of eleven spawns in each aquarium were incubated to determine hatching success. Unincubated eggs were pooled by treatment levels, placed in separate glass vials and stored frozen for possible analysis.. Eggs spawned by control fish were incubated in aquaria where no first generation fish survived or where spawning was reduced or absent. Twenty fry from each of the first two successfully hatched egg groups in each aquarium were impartially selected and placed in their respective growth chambers. After 34 days exposure, each fry group was photographed to determine percentage survival and total length. Each group was wet weighed and the average individual weight calculated. Each group was stored frozen in a glass vial for possible analysis.

- Exposure of first generation fish was terminated on test day 283 when no spawning had occurred in any aquarium for a period of one week. Each fish was individually measured with a centimeter ruler for total length, wet weight and internally examined to verify sex and gonadal condition. Males and females in each aquarium were separated and frozen as whole fish in glass jars for possible residue analysis.

RANGE-FINDING STUDY
- Test concentrations: Negative control, solvent control, 3.1, 6.2, 12, 25 and 50 µg/L.
- Results used to determine the conditions for the definitive study: Mortality of fry during this exposure occurred during the first 96 hours and remained relatively unchanged for the remainder of the 35 days. The 35-day LC50 and 95% confidence interval were calculated to be 23 (19 - 26 ) µg/L. Based on the response of fry during the preliminary exposure, the nominal concentrations selected for the chronic study were 1.5, 3.1, 6.2, 12 and 25 µg/L.

Reference substance (positive control):

no

Key result

Duration:

297 d

Dose descriptor:

NOEC

Effect conc.:

1.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

number of eggs per surviving female per day

Remarks on result:

other: Calculated in a separate study (Yellowlees, A. 2016 )

Duration:

297 d

Dose descriptor:

NOEC

Effect conc.:

3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

F0 and F1 generation

Details on results:

An overview of the results is provided in Table 2 - Table 7 in 'Any other information on results incl. tables'

FIRST GENERATION (F0)
- Hatching success of eggs was significantly reduced by exposure to a mean measured concentration of 16 µg/L the test substance when compared to the control and solvent control. Thirty days after hatching, survival of F0 fry exposed to 16 µg/L was significantly reduced as compared to survival of both control groups. Only seven F0 fry exposed to 16 µg/L survived, with nearly all mortality occuring in the first week of exposure. Growth ( total length) of fry was similar in all test concentrations ≤ 16 µg/L and controls. After 64 days exposure, survival of F0 fry remained unaffected by exposure to concentrations ≤ 6.5 µg/L.
- Statistically significant differences were indicated when the mean total lengths of F0 fry , exposed to 1.4 and 6.5 µg/L the test substance and acetone, were compared to control. However, these differences were actually less than one millimeter which was below the accuracy of the measuring technique. These responses did not appear to be toxicant related and on day 283 when F0 fish were terminated, there were no differences in total lengths between these fish and controls.
- All F0 fry in the A replicate aquarium of solvent control died between day 16 and 33. This isolated incident of mortality among fish exposed to acetone was attributed to a filamentous growth that occurred in the aquarium during this time. Frequent brushing, syphoning and rinsing of the larval chambers failed to control this microbial growth. Fry appeared to be severely stressed and were observed to have filaments of the growth trailing from their gills and bodies. On day 64, fifteen fish were transferred to this aquarium from the B replicate of solvent control and provided data for the remainder of the study.
- On test day 168, a total of five male fish in four different aquaria were diagnosed to have gas bubble disease. The temperature of the unheated water entering the aquaria was 10°C lower than
the temperature of the water in the aquaria. It was determined that the diluent water should be heated before entering the aquaria to eliminate the sudden decrease in temperature and gas pressure in the aquaria water. For the remainder of the study, diluent water was delivered to the chronic unit through a heated water system, and no additional cases of gas bubble disease occurred.
- On test day 172, fish in each aquarium were examined to determine their sex and establish a sex ratio of three males to six females wherever possible. In four aquaria, fewer than six females were present to select. The male fish previously diagnosed to have gas bubble disease and still exhibiting symptoms were removed at this time as extra males.
- The survival of fish exposed for 283 days to concentrations ≤16 µg/L remained relatively unchanged from that observed on day 35. Analysis of variance of individual total lengths and wet weights of male and female fish indicated no differences in growth between fish exposed to test concentrations ≤16 µg/L the test substance and control or solvent control fish.

SPAWNING
- The number of eggs per spawns was significantly less for fish exposed to 16 and 6.5 µg/L when compared to control and solvent control spawns.

SECOND GENERATION (F1)
- No eggs hatched successfully when transferred from control and incubated in 16 µg/L treatment. Hatching success of eggs spawned by F0 fish exposed to 6.5 µg/L was significantly reduced when compared to control. Statistical analysis also indicated reduced hatching success for eggs spawned by solvent control fish. However, the hatching failure of several early, and possibly infertile, spawns in the A replicate aquarium greatly reduce the mean percentage hatch for the treatment. Hatchability of eggs spawned in the B replicate aquarium of solvent control was comparable to control. After 34 days exposure, survival, total length and wet weight of F1 fry was unaffected by
test concentrations ≤ 6.5 µg/L.

Reported statistics and error estimates:

Data for survival, growth and reproduction were subjected to a one-way analysis of variance (P = 0.05) (Steel and Torrie, 1968). Percentage survival and percentage hatching success were transformed to arc sin √percentage prior to analysis of variance. If treatment effects was indicated, the treatment means for that parameter were compared to the control and to the solvent control mean by Dunnett ’ s procedure using one-sided comparison (Steel and Torrie, 1960).

Table 2. Hatching success, survival and total length of F0 fathead minnows after 35 and 64 days exposure to the test substance

Mean measured concentration (µg/L)	replicate	Hatching success (%)	Day 35		Day 64
			Survival (%)	Total length ± S.D. (mm)	Survival (%)	Total length ± S.D. (mm)
Control	A	95	93	20 ± 2	88	29 ± 2
	B	85	95	21 ±3	95	28 ± 4
Solvent control	A	92	0^e	-	-	-
	B	93	98	20 ± 3	95	27 ± 5
0.60	A	96	100	20 ± 2	100	27 ± 3
	B	96	98	20 ± 2	98	28 ± 3
1.4	A	83	100	20 ± 2	93	27 ± 3
	B	98	100	19 ± 3	100^d	27 ± 4
3.0	A	82	93	20 ± 3	88^c	27 ± 3
	B	96	95	20 ± 3	95^c	28 ± 4
6.5	A	74	90	20 ± 3	88	27 ± 3
	B	85	98	20 ± 3	95	27 ± 3
16	A	42^a	8^a	21 ± 3^b	8	29 ± 5
	B	51^a	10^a	-	-	-

a Significantly (P=0.05) different from control.

b Fish from both replicates were combined.

c One fish with distended opercula.

d Two fish with distended opercula.

e All fish in replicate A of acetone control died between test days 16 and 33.

Table 3. Survival and sex ratio of F0 fathead minnows exposed 172 days to the test substance.

Mean measured concentration (µg/L)	replicate	# of mortalities between days 64 - 172	Sex ratio Male/Female	Total # fish	Retained as spawners male/Female	Total # fish
Control	A	1	8^f/6	14	3/6	9
	B	2	5/8	13	3/6	9
Solvent control	A	0	9/6	15	3/6	9
	B	0	8/7	15	3/6	9
0.60	A	0	8^d/7	15	3/6	9
	B	2	6^d/7	13	3/6	9
1.4	A	1	9^c/5	14	3/5	8
	B	0	7/8	15	3/6	9
3.0	A	2	9^b/4	13	3/4	7
	B	3	8/4	12	3/4	7
6.5	A	1	7^a/7	14	3/6	9
	B	0	6/9	15	3/6	9
16	A	1	3/2	5	3/2	5
	B	-	-	-	-	-

a One fish with scoliosis, one with gas bubble disease.

b One fish with gas bubble disease.

c Two fish with protruding eyes probably due to gas bubble disease.

d One fish with distended operculum.

e On day 64, 15 fish were transferred to this aquarium from the B replicate aquarium of acetone control.

f Two fish with gas bubble disease.

Table 4. Survival, growth and sexual maturity of F0 fathead minnows after 283 days exposure to the test substance.

Mean measured concentration (µg/L)	replicate	Number of survivors		Number of mortalities (day 173 - 283)	Total length (mm) ± S.D.		Total weight (g) ± S.D.
		male	female		male	female	male	female
Control	A	4	5	0	76± 6	55 ± 3	4.64 ± 1.09	1.42 ± 0.21
	B	3	6	0	77± 4	55 ± 6	4.55 ± 0.68	1.37 ± 0.40
Solvent control	A	3	6	0	81± 5	58 ± 5	5.46 ± 0.89	1.74 ± 0.57
	B	3	6	0	84 ± 5	56 ± 5	6.09 ± 1.46	1.58 ± 0.45
0.60	A	3	6	0	78± 2	58 ± 4	4.80 ± 0.91	1.72 ± 0.35
	B	3	6	0	81± 7	54 ± 4	5.46 ± 1.26	1.34 ± 0.32
1.4	A	4	3	1	80± 2	55 ± 4	5.04 ± 0.72	1.35 ± 0.35
	B	3	6	0	80± 6	55 ± 5	5.32 ± 1.21	1.41 ± 0.44
3.0	A	3	4	0	79± 8	60 ± 4	4.88 ± 1.18	1.66 ± 0.43
	B	4	3	0	78± 3	63 ± 5	4.42 ± 0.77	1.94 ± 0.15
6.5	A	3	6	0	82± 10	56 ± 3	5.30 ± 2.68	1.45 ± 0.23
	B	3	6	0	81± 2	57 ± 3	5.27 ± 0.38	1.60 ± 0.33
16	A	3	2	0	82 ± 1	58 ± 1	5.15 ± 0.38	1.55 ± 0.06
	B	-	-	-	-	-	-	-

 

Table 5. Reproductive success of fathead minnows chronically exposed to the test substance

Mean measured concentration (µg/L)	replicate	Total spawns	Total eggs	Eggs/spawn
Control	A	31	2265	73
	B	30	3660	122
Solvent control	A	18	2332	130
	B	14	1339	96
0.60	A	20	3564	178
	B	5	301	60
1.4	A	18	1868	104
	B	30	3266	109
3.0	A	6	713	119
	B	2	111	56
6.5	A	1	12	12^a
	B	14	676	48^a
16	A	1	18	18^a
	B	-	-	-

a Significantly different (P=0.05) from control.

  Table 6. Hatching success, survival and growth of F1 fathead minnows exposed to the test substance for 34 days.

Mean measured concentration (µg/L)	replicate	Hatching success mean ± S.D. (%)	Number of egg groups incubated	Survival (%)	Total length (mm)	Average wet weight (g)
Control	A	94 ± 3	10	88	25 ± 3	0.1218
	B	92 ± 4	11	90	23 ± 3	0.0973
Solvent control	A	51^b± 41	11	95	23 ± 3	0.1137
	B	85 ±11	6	98	24 ± 3	0.1073
0.60	A	84± 30	10	88	23 ± 2	0.0866
	B	82± 8	2	95	24 ± 2	0.1052
1.4	A	84± 13	10	100	24 ± 2	0.1188
	B	74± 37	11	95	21 ± 3	0.0772
3.0	A	95± 2	4	92	24 ± 2	0.0998
	B	62± 26	2	100	24 ± 3	0.1186
6.5	A	60± 38	4^a	78	24 ± 2	0.1095
	B	40^b± 26	3^a	80	22 ± 3	0.0847
16	A	0	3^a	-	-	-
	B	0	3	-	-	-

a Eggs and subsequent fry transferred from control.

b Significantly different from control.

Table 7. Comparison of hatchability of F1 eggs produced by parental fish exposed to the test substance with F1 eggs produced by unexposed parents (control fish).

Mean measured concentration (µg/L)	Mean percentage hatching success
	Eggs from exposed parents	Number of egg groups incubated	Eggs from unexposed parents^a	Number of egg groups incubated
3.0	62	2	90	3
6.5	40^b	3	88	3

a Eggs transferred from control spawns to treatment levels.

b Significantly (P=0.05) lower than hatching success of eggs from unexposed parents.

Validity criteria fulfilled:

not specified

Conclusions:

Based on the findings, the NOEC was determinded to be 3.0 µg/L for reproduction endpoint (both F0 and F1 generations). The raw data were re-analysed in a separate study. It concluded that the NOEC value for number of eggs per surviving female per day was 1.4 µg/L.

Executive summary:

An early life-stage test was performed with Pimephales promelas, and extended over two generations (in total 297 days of exposure). This study was conduced without following guideline, but in according to EPA “Recommended bioassay procedures for fathead minnow (Pimephales promelas, Rafinesque) chronic tests” (1971) and in compliance with GLP criteria. The test organism was exposed to the test substance (96% a.i) in five nominal concentrations, 1.5, 3.1, 6.2, 12.5, and 25 µg/L (measured concentration 0.6, 1.4, 3.0, 6.5 and 16 µg/L, respectively; measured byGC with Ni-electron capture detector). In addition, solvent control (acetone, max. 0.005 mL/L) and control were also included in the test. A stability analysis indicates that in water after 3 days 34% of the material remained, and that the acetone stock solutions were stable for 15 days. Actual concentrations in the aquaria ranged from 40 - 64% of nominal. Actual concentrations in the delivery tubes are 2.2 (sd. 1.0, n = 14) times higher than in the aquaria. The low actual concentrations are probably due to adsorption to feed, excreta, algae, fish, glass, and/or biotransformation.

Hatching success of eggs and survival of first generation (F0) fry after 30 days exposure were significantly reduced by exposure to a mean measured concentration of 16 µg/L the test substance. Reproductive success of F0 fish was adversely affected by exposure to concentrations ≥ 6.5 µg/L, which significantly reduced the number of eggs per spawn, Hatching success of second generation (F1) eggs was significantly reduced by exposure to 6.5 µg/L.

Based on the findings, the NOEC of reproduction (both F0 and F1 generations) was determined to be 3.0 µg/L. The raw data were re-analysed in a separate study. It concluded that the NOEC value for number of eggs per surviving female per day was 1.4 µg/L.

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

7 Aug 2013 to 9 Sep 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

2012

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Draft, 1996

Principles of method if other than guideline:

Besides the guidelines, the study was conducted also according to ASTM E: 1241 - 05 (2013).

GLP compliance:

yes

Test organisms (species):

Cyprinodon variegatus

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Key result

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

11 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

mean dry weight

Analytical data

The concentrations of the test substance were determined in the test solutions. The mean measured concentrations ranged from 68 to 80% of their nominal concentrations. The limit of quantification was 0.0268 μg/L. The mean measured concentrations were used for calculating and reporting the results.

 Table 1. Analytical results

Nominal concentration (µg/L)	Measured concentration (µg/L)							% CV	Percent nominal (%)
	Day 0	Day 5	Day 13	Day 16	Day 20	Day 33	Mean (SD)
Control	<0.10	<0.11	<0.10	<0.10	<0.10	<0.10	NA	NA	NA
1.9	1.4	1.4	1.2	1.2	1.2	1.2	1.3 (0.12)	9.1	68
3.8	3.4	3.2	2.9	2.9	2.7	3.2	3.0 (0.27)	8.8	80
7.5	6.3	5.2	6.1	4.6	4.8	5.4	5.4 (0.69)	13	72
15	11	13	11	9.6	8.8	10	11 (1.4)	13	70
30	25	24	24	25	22	24	24 (0.98)	4.1	80

Biological data (an overview of the results is provided in Table 2 below)

At exposure initiation, a subsample of 30 embryos was collected and the embryonic stage of development was determined. The median was 14 (one half epiboly), the mean was 11 (flat blastula) and range of the developmental stages was from 3 to 14 (encompassing one-celled blastodisc to one half epiboly).

On day 5, all viable embryos in the negative control were hatched. Completion of hatch for all treatment levels was also day 5. No delays to completion of hatch (time to hatch) were observed for any treatment level compared to the negative control.

At the completion of the hatching period (day 5), hatching success in the negative control averaged 82%. Embryo hatching success in the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels averaged 84, 88, 84, 89 and 88%, respectively. Statistical analysis (Fisher’s Exact Test with Bonferroni-Holm Adjustment) determined no significant difference in hatching success among embryos exposed to any of the treatment levels tested, compared to the negative control (82%). The NOEC for this endpoint was determined to be 24 µg/L.

At the completion of the hatching period (day 5), the mean percent of live, normal larvae in the negative control averaged 100%. Percent of live, normal larvae in the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels all averaged 100%. Statistical analysis (Fisher’s Exact Test with Bonferroni-Holm Adjustment) determined no significant difference in percent of live, normal larvae among embryos exposed to any of the treatment levels tested, compared to the negative control (100%). The NOEC for this endpoint was determined to be 24 µg/L.

At exposure termination (33 days post-hatch, 28 days post-hatch-completion), larval survival in the negative control averaged 96%. Larval survival in the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels averaged 96, 94, 98, 89 and 94%, respectively. Statistical analysis (Fisher’s Exact Test with Bonferroni-Holm Adjustment) determined no significant difference in larval survival among embryos exposed in any of the treatment levels tested, compared to the negative control (96%). The NOEC for this endpoint was determined to be 24 µg/L.

The mean total length of larvae exposed to the negative control averaged 19.8 mm. The mean total length of larvae exposed to the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels was 20.0, 19.5, 19.3, 19.6 and 18.6 mm, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined a significant reduction in mean total length of larvae exposed to the 24 µg/L treatment levels compared to the negative control (19.8 mm). The NOEC for this endpoint was determined to be 11 µg/L.

The mean wet weight of larvae exposed to the negative control averaged 0.1155 g. The mean wet weight of larvae exposed to the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels was 0.1156, 0.1123, 0.1060, 0.1082 and 0.0976 g, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined a significant reduction in mean wet weight of larvae exposed to the 24 µg/L treatmentlevel, compared to the negative control (0.1155 g). The NOEC for this endpoint was determined to be 11µg/L. At exposure termination, the mean dry weight of larvae exposed to the negative control averaged 0.0282 g. The mean dry weight of larvae exposed to the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels was 0.0283, 0.0276, 0.0254, 0.0272, 0.0242 g, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined a significant difference in mean wet weight of larvae exposed to the 5.4 and 24µg/L treatment levels, compared to the negative control (0.0282 g). However, the reduction in the 5.4 µg/L treatment level is not considered to be treatment-related due to the lack of a clear dose response. The NOEC for this endpoint was determined to be 11 µg/L. The biological performance of the negative control organisms exceeded the minimum acceptability criterion outlined in the study protocol as the average hatchability of the negative control was above 75% and the average survival (post-hatch) of larvae in the negative control was above 80%. The dissolved oxygen concentration was > 60% of the air saturation value during the test (i.e. > 83.5%).

 

Table 2.Effects of the test substance on the growth of sheepshead minnow (Cyprinodon variegatus)

Mean measured concentration (µg ai/L)	Hatching success (%)	Live, Normal Larvae at Hatch (%)	Larval survival (%)	Mean length (mm) ± SD	Mean wet weight (g) ± SD	Mean dry weight (g) ± SD
Control	82	100	96	19.8 (0.20)	0.1155 (0.0057)	0.0282 (0.0011)
1.3	84	100	96	20.0 (0.38)	0.1156 (0.0051)	0.0283 (0.0014)
3.0	88	100	94	19.5 (0.59)	0.1123 (0.0112)	0.0276 (0.0024)
5.4	84	100	98	19.3 (0.28)	0.1060 (0.0060)	0.0254 (0.0006)#
11	89	100	89	19.6 (0.10)	0.1082 (0.0037)	0.0272 (0.0011)
24	88	100	94	18.6 (0.41)*	0.0976 (0.0036)*	0.0242 (0.0011)*

# Significantly reduced compared to the control, based on Dunnett’s Multiple Comparison Test. This reduction is not considered to be treatment-related due to the lack of a clear dose response.

* Significantly reduced compared to the control, based on Dunnett’s Multiple Comparison Test.

Validity criteria fulfilled:

yes

Conclusions:

Toxicity endpoints identified for the test substance to early life stages of sheepshead minnow were determined. Based on mean measured concentrations and all endpoints examined for the exposure, the NOEC was determined to be 11 µg/L.

Executive summary:

This toxicity study was performed to determine the chronic toxicity of the test substance to sheepshead minnow (Cyprinodon variegatus) embryos, larvae and fry under flow-through conditions following OCSPP (Draft) Guideline 850.1400 and OECD TG 210. The study was conducted in compliance with GLP criteria. Four replicate exposure aquaria, each containing 20 embryos, were established for each exposure and control. The test organisms were exposed to nominal concentrations of 1.9, 3.8, 7.5, 15 and 30 µg/L (1.3, 3.0, 5.4, 11 and 24 µg/L, respectively mean measured concentration using GC). Exposure solution concentrations were analytically confirmed on days 0, 5, 13, 20, 27 and 33. Results of this study are based on mean measured concentrations. The test was conducted using an exposure system consisting of an intermittent-flow proportional diluter, a temperature-controlled water bath and a set of 24 exposure aquaria. The exposure system was designed to provide five concentrations of the test substance and a dilution water (negative) control to four replicate exposure aquaria per treatment. The exposure system maintained a temperature range of 25 - 27 °C and a photoperiod of 16 hours of light with a light intensity range of 50 to 54 footcandles (540 to 580 lux) and 8 hours of dark. Embryo hatching success, percentage of embryos that produce live, normal larvae at hatch, percentage of surviving larvae at test termination and total length and wet and dry weight values were used to determine the NOEC and the LOEC.

On day 5, all viable embryos in the negative control were hatched. Completion of hatch for all treatment levels was also day 5. No delays to completion of hatch (time to hatch) were observed for any treatment level compared to the negative control. At the completion of hatch (day 5), hatching success in the negative control averaged 82%. Embryo hatching success in the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels averaged 84, 88, 84, 89 and 88%, respectively. Statistical analysis (Fisher’s Exact Test with Bonferroni−Holm Adjustment) determined no significant difference in hatching success among embryos exposed to any of the treatment levels tested, compared to the negative control (82%). The NOEC and LOEC for this endpoint were determined to be 24 and > 24 µg/L, respectively. At the completion of hatch (day 5), the percent of live, normal larvae in the negative control averaged 100%. Percent of live, normal larvae in the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels all averaged 100%. The NOEC and LOEC for this endpoint were determined to be 24 and > 24 µg/L, respectively. At exposure termination (33 days post-hatch, 28 days post-hatch-completion), larval survival in the negative control averaged 96%. Larval survival in the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels averaged 96, 93, 98, 90 and 95%, respectively. The NOEC and LOEC for this endpoint were determined to be 24 and > 24 µg/L, respectively. At exposure termination (33 days post-hatch, 28 days post-hatch-completion), the mean total length of larvae exposed to the negative control averaged 19.8 mm. The mean total length of larvae exposed to the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels was 20.0, 19.5, 19.3, 19.6 and 18.6 mm, respectively. The NOEC and LOEC for this endpoint were determined to be 11 and 24 µg/L, respectively. At exposure termination (33 days post-hatch, 28 days post-hatch-completion), the mean wet weight of larvae exposed to the negative control averaged 0.1155 g. The mean wet weight of larvae exposed to the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels was 0.1156, 0.1123, 0.1060, 0.1082, 0.0976 g, respectively. The NOEC and LOEC for this endpoint were determined to be 11 and 24 µg/L, respectively. At exposure termination, the mean dry weight of larvae exposed to the negative control averaged 0.0282 g. The mean dry weight of larvae exposed to the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels was 0.0283, 0.0276, 0.0254, 0.0272, 0.0242 g, respectively. The NOEC and LOEC for this endpoint were determined to be 11 and 24 µg/L, respectively. The biological performance of the negative control organisms exceeded the minimum guideline acceptability criterion outlined in the study protocol as the average hatchability of the negative control was > 75% and the average survival (post-hatch) of larvae in the negative control was > 80%.

Based on mean measured concentrations and mean dry weight, the most sensitive endpoint, the NOEC was determined to be 11 µg/L.

Description of key information

Freshwater, 297-d NOEC = 1.4 µg/L, Pimephales promelas, reproduction, no guideline followed, Shults 1980

Marine water, 33-d NOEC = 11 µg/L, Cyprinodon variegatus, dry weight, OECD TG 201, Schwader 2014

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

1.4 µg/L

Marine water fish

Marine water fish

Dose descriptor:

NOEC

Effect concentration:

11 µg/L

Additional information

Freshwater

An early life-stage test was performed with Pimephales promelas, and extended over two generations (in total 297 days of exposure). This study was conducted without following guideline, but in according to EPA “Recommended bioassay procedures for fathead minnow (Pimephales promelas, Rafinesque) chronic tests” (1971) and in compliance with GLP criteria. The test organism was exposed to the test substance (96% a.i) in five nominal concentrations, 1.5, 3.1, 6.2, 12.5, and 25 µg/L (measured concentration 0.6, 1.4, 3.0, 6.5 and 16 µg/L, respectively; measured by GC with Ni-electron capture detector). In addition, solvent control (acetone, max. 0.005 ml/l) and control were also included in the test. A stability analysis indicates that in water after 3 days 34% of the material remained, and that the acetone stock solutions were stable for 15 days. Actual concentrations in the aquaria ranged from 40 - 64% of nominal. Actual concentrations in the delivery tubes are 2.2 (sd. 1.0, n = 14) times higher than in the aquaria. The low actual concentrations are probably due to adsorption to feed, excreta, algae, fish, glass, and/or biotransformation.

Hatching success of eggs and survival of first generation (F0) fry after 30 days exposure were significantly reduced by exposure to a mean measured concentration of 16 µg/L the test substance. Reproductive success of F0 fish was adversely affected by exposure to concentrations ≥ 6.5 µg/L, which significantly reduced the number of eggs per spawn, Hatching success of second generation (F1) eggs was significantly reduced by exposure to 6.5 µg/L.

Based on the findings, the NOEC of reproduction (both F0 and F1 generations) was determined to be 3.0 µg/L. The raw data were re-analysed in a separate study. It concluded that the NOEC value for number of eggs per surviving female per day was 1.4 µg/L.

Marine water

This toxicity study was performed to determine the chronic toxicity of the test substance to sheepshead minnow (Cyprinodon variegatus) embryos, larvae and fry under flow-through conditions following OCSPP (Draft) Guideline 850.1400 and OECD TG 210. The study was conducted in compliance with GLP criteria. Four replicate exposure aquaria, each containing 20 embryos, were established for each exposure and control. The test organisms were exposed to nominal concentrations of 1.9, 3.8, 7.5, 15 and 30 µg/L (1.3, 3.0, 5.4, 11 and 24 µg/L, respectively mean measured concentration using GC). Exposure solution concentrations were analytically confirmed on days 0, 5, 13, 20, 27 and 33. Results of this study are based on mean measured concentrations. The test was conducted using an exposure system consisting of an intermittent-flow proportional diluter, a temperature-controlled water bath and a set of 24 exposure aquaria. The exposure system was designed to provide five concentrations of the test substance and a dilution water (negative) control to four replicate exposure aquaria per treatment. The exposure system maintained a temperature range of 25 - 27 °C and a photoperiod of 16 hours of light with a light intensity range of 50 to 54 footcandles (540 to 580 lux) and 8 hours of dark. Embryo hatching success, percentage of embryos that produce live, normal larvae at hatch, percentage of surviving larvae at test termination and total length and wet and dry weight values were used to determine the NOEC and LOEC.

On day 5, all viable embryos in the negative control were hatched. Completion of hatch for all treatment levels was also day 5. No delays to completion of hatch (time to hatch) were observed for any treatment level compared to the negative control. At the completion of hatch (day 5), hatching success in the negative control averaged 82%. Embryo hatching success in the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels averaged 84, 88, 84, 89 and 88%, respectively. Statistical analysis (Fisher’s Exact Test with Bonferroni−Holm Adjustment) determined no significant difference in hatching success among embryos exposed to any of the treatment levels tested, compared to the negative control (82%). The NOEC and LOEC for this endpoint were determined to be 24 and > 24 µg/L, respectively. At the completion of hatch (day 5), the percent of live, normal larvae in the negative control averaged 100%. Percent of live, normal larvae in the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels all averaged 100%. The NOEC and LOEC for this endpoint were determined to be 24 and > 24 µg/L, respectively. At exposure termination (33 days post-hatch, 28 days post-hatch-completion), larval survival in the negative control averaged 96%. Larval survival in the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels averaged 96, 93, 98, 90 and 95%, respectively. The NOEC and LOEC for this endpoint were determined to be 24 and > 24 µg/L, respectively. At exposure termination (33 days post-hatch, 28 days post-hatch-completion), the mean total length of larvae exposed to the negative control averaged 19.8 mm. The mean total length of larvae exposed to the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels was 20.0, 19.5, 19.3, 19.6 and 18.6 mm, respectively. The NOEC and LOEC for this endpoint were determined to be 11 and 24 µg/L, respectively. At exposure termination (33 days post-hatch, 28 days post-hatch-completion), the mean wet weight of larvae exposed to the negative control averaged 0.1155 g. The mean wet weight of larvae exposed to the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels was 0.1156, 0.1123, 0.1060, 0.1082, 0.0976 g, respectively. The NOEC and LOEC for this endpoint were determined to be 11 and 24 µg/L, respectively. At exposure termination, the mean dry weight of larvae exposed to the negative control averaged 0.0282 g. The mean dry weight of larvae exposed to the 1.3, 3.0, 5.4, 11 and 24 µg/L treatment levels was 0.0283, 0.0276, 0.0254, 0.0272, 0.0242 g, respectively. The NOEC and LOEC for this endpoint were determined to be 11 and 24 µg/L, respectively. The biological performance of the negative control organisms exceeded the minimum guideline acceptability criterion outlined in the study protocol as the average hatchability of the negative control was > 75% and the average survival (post-hatch) of larvae in the negative control was > 80%.

Based on mean measured concentrations and mean dry weight, the most sensitive endpoint, the NOEC was determined to be 11 µg/L.